Indoles as NAALADase inhibitors

ABSTRACT

This invention relates to new compounds, pharmaceutical compositions and diagnostic kits comprising such compounds, and methods of using such compounds for inhibiting NAALADase enzyme activity, detecting diseases where NAALAdase levels are altered, effecting neuronal activity, effecting TGF-β activity, inhibiting angiogenesis, and treating glutamate abnormalities, neuropathy, pain, compulsive disorders, prostate diseases, cancers and glaucoma.

This invention relates to new compounds, pharmaceutical compositions anddiagnostic kits comprising such compounds, and methods of using suchcompounds for inhibiting NAALADase enzyme activity, detecting diseaseswhere NAALADase levels are altered, effecting neuronal activity,effecting TGF-β activity, inhibiting angiogenesis, and treatingglutamate abnormalities, neuropathy, pain, compulsive disorders,prostate diseases, cancers, glaucoma and retinal disorders.

The NAALADase enzyme, also known as prostate specific membrane antigen(“PSM” or “PSMA”) and human glutamate carboxypeptidase II (“GCP II”),catalyzes the hydrolysis of the neuropeptide N-acetyl-aspartyl-glutamate(“NAAG”) to N-acetyl-aspartate (“NAA”) and glutamate. Based upon aminoacid sequence homology, NAALADase has been assigned to the M28 family ofpeptidases.

Studies suggest that NAALADase inhibitors may be effective in treatingischemia, spinal cord injury, demyelinating diseases, Parkinson'sdisease, Amyotrophic Lateral Sclerosis (“ALS”), alcohol dependence,nicotine dependence, cocaine dependence, cancer, neuropathy, pain andschizophrenia, and in inhibiting angiogenesis. In view of their broadrange of potential applications, a need exists for new NAALADaseinhibitors and pharmaceutical compositions comprising such compounds.

SUMMARY OF THE INVENTION

This invention relates to a compound of formula I

or a pharmaceutically acceptable equivalent of said compound, wherein:

A¹, A², A³ and A⁴ are independently hydrogen, C₁–C₉ alkyl, C₂–C₉alkenyl, C₂–C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle, C₁–C₉alkoxy, C₂–C₉ alkenyloxy, phenoxy, benzyloxy, hydroxy, halo, nitro,cyano, isocyano, —COOR⁶, —COR⁶, —NR⁶R⁷, —SR⁶, —SOR⁶, —SO₂R⁶, —SO₂(OR⁶),—(C═O)NR⁶R⁷, —(C═O)NR⁶(CH₂)_(n)COOH, —NR⁶(C═O)R⁷ or —(CH₂)_(n)COOH, orany adjacent two of A¹, A², A³ and A⁴ form with the benzene ring a fusedring that is saturated or unsaturated, aromatic or non-aromatic, andcarbocyclic or heterocyclic, said heterocyclic ring containing 1 or 2oxygen, nitrogen and/or sulfur heteroatom(s);

n is 1–3;

R, R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are independently hydrogen, carboxy,C₁–C₉ alkyl, C₂–C₉ alkenyl, C₂–C₉ alkynyl, aryl, heteroaryl, carbocycleor heterocycle; and

said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle,alkoxy, alkenyloxy, phenoxy, benzyloxy and fused ring are independentlyunsubstituted or substituted with one or more substituent(s).

Additionally, this invention relates to a method for treating aglutamate abnormality, treating a compulsive disorder, effecting aneuronal activity, treating a prostate disease, treating cancer,inhibiting angiogenesis, or effecting a TGF-β activity, comprisingadministering to a mammal in need of such inhibition, treatment oreffect, an effective amount of a compound of formula I, as describedabove.

This invention further relates to a method for detecting a disease,disorder or condition where NAALADase levels are altered, comprising:

(i) contacting a sample of bodily tissue or fluid with a compound offormula I, as defined above, wherein said compound binds to anyNAALADase in said sample; and

(ii) measuring the amount of any NAALADase bound to said sample, whereinthe amount of NAALADase is diagnostic for said disease, disorder orcondition.

This invention also relates to a method for detecting a disease,disorder or condition where NAALADase levels are altered in an animal ora mammal, comprising:

(i) labeling a compound of formula I, as defined above, with an imagingreagent;

(ii) administering to said animal or mammal an effective amount of thelabeled compound;

(iii) allowing said labeled compound to localize and bind to NAALADasepresent in said animal or mammal; and

(iv) measuring the amount of NAALADase bound to said labeled compound,wherein the amount of NAALADase is diagnostic for said disease, disorderor condition.

Additionally, this invention further relates to a diagnostic kit fordetecting a disease, disorder or condition where NAALADase levels arealtered, comprising a compound of formula I, as defined above, labeledwith a marker.

Finally, this invention relates to a pharmaceutical compositioncomprising:

(i) an effective amount of a compound of formula I, as described above;and

(ii) a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a bar graph showing the effect of2-(phosphonomethyl)pentanedioic acid (“Compound C”) on TGF-β1concentrations in ischemic cell cultures.

FIG. 2 is a bar graph showing the effect of Compound C on TGF-β2concentrations in ischemic cell cultures.

FIG. 3 is a bar graph showing the reversal of the neuroprotective effectof Compound C by TGF-β neutralizing antibodies in ischemic cellcultures.

FIG. 4 is a bar graph showing the non-reversal of the neuroprotectiveeffect of Compound C by FGF neutralizing antibodies in ischemic cellcultures.

FIG. 5 is a bar graph showing the reversal of the neuroprotective effectof Compound C by TGF-β neutralizing antibodies in rats subjected tomiddle cerebral artery occlusion (“MCAO”).

FIG. 6 is a bar graph showing the effect of Compound C on TGF-β1 levelsduring occlusion and reperfusion in rats subjected to MCAO.

FIG. 7A is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle or2-[[2,3,4,5,6-pentafluorobenzyl)hydroxyphosphinyl]methyl]pentanedioicacid (“Compound A”), against the days following administration with STZ.

FIG. 7B is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle or2-(2-sulfanylethyl)pentanedioic acid (“Compound D”), against the daysfollowing administration with STZ.

FIG. 8 is a bar graph plotting the withdrawal latency difference scoresof normal (unoperated) rats and chronic constrictive injury-induced ratstreated with a vehicle or Compound C, against the days followingsurgery.

FIG. 9A is a bar graph plotting the motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound A, against the weeks post STZ.

FIG. 9B is a bar graph plotting the sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound A, against the weeks post STZ.

FIG. 10A is a bar graph plotting the motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D, against the weeks post STZ.

FIG. 10B is a bar graph plotting the sensory nerve conduction velocityof non-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D, against the weeks post STZ.

FIG. 11 is a graph plotting the withdrawal latency of non-diabetic ratsand BB/W diabetic rats treated with a vehicle, Compound D or Compound A,against the weeks of treatment.

FIG. 12 is a graph plotting the nerve conduction velocity ofnon-diabetic rats and BB/W diabetic rats treated with a vehicle,Compound D or Compound A, against the weeks of treatment.

FIG. 13 is a bar graph plotting the percent of transgenic mice at 210days of age that exhibited limb shaking after treatment with2-(3-sulfanylpropyl)pentanedioic acid (“Compound B”) or a vehicle.

FIG. 14 is a bar graph plotting the gait, measured on an arbitrary scaleranging from 0 to 3, of transgenic mice at 210 days of age aftertreatment with Compound B or a vehicle.

FIG. 15 is a bar graph plotting hind limbs dragging, measured on anarbitrary scale ranging from 0 to 3, of transgenic mice at 210 days ofage after treatment with Compound B or a vehicle.

FIG. 16 is a bar graph plotting the crossing of limbs, measured on anarbitrary scale ranging from 0 to 3, of transgenic mice at 210 days ofage after treatment with Compound B or a vehicle.

FIG. 17 is a bar graph plotting the righting reflex of transgenic mice,measured by the time (seconds) it took the mice to right themselves whenplaced on their sides, at 210 days of age after treatment with CompoundB or a vehicle.

FIG. 18 is a graph plotting the percent of transgenic mice treated withCompound B or a vehicle that died against the age of the mice (days).

FIG. 19 is a Kaplan-Meier survival graph plotting the percent oftransgenic mice treated with Compound B or a vehicle that survivedagainst the number of days that the mice were on study therapy.

FIG. 20 is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle,Compound D or 3-carboxy-alpha-(3-mercaptopropyl)benzenepropanoic acid(“Compound E”), against the weeks of treatment.

FIG. 21 is a bar graph plotting motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle, CompoundD or Compound E, against the weeks of treatment.

FIG. 22 is a bar graph plotting sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle, CompoundD or Compound E, against the weeks of treatment, where treatment started5 weeks post STZ.

FIG. 23 is a bar graph plotting the withdrawal latency difference scoresof non-diabetic rats and STZ-diabetic rats treated with a vehicle orlower doses of Compound D (1 and 3 mg/kg), against the weeks oftreatment, where treatment started 7 weeks post STZ.

FIG. 24 is a bar graph plotting motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle or lowerdoses of Compound D (1 and 3 mg/kg), against the weeks of treatment,where treatment started 7 weeks post STZ.

FIG. 25 is a bar graph plotting sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle or lowerdoses of Compound D (1 and 3 mg/kg), against the weeks of treatment,where treatment started 7 weeks post STZ.

FIG. 26 are bar graphs plotting sensory nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D at 35 days and 60 days after treatment, where treatmentstarted 60 days post STZ.

FIG. 27 are bar graphs plotting motor nerve conduction velocity ofnon-diabetic rats and STZ-diabetic rats treated with a vehicle orCompound D at 35 days after treatment, where treatment started 60 dayspost STZ.

FIG. 28 is a graph plotting sensory nerve conduction velocity ofnon-diabetic and STZ-diabetic rats treated with a vehicle or Compound D,against the days post STZ, where treatment started 90 days post STZ.

FIG. 29 is a bar graph plotting motor and sensory nerve conductionvelocities of non-diabetic mice and db/db diabetic mice before treatmentwith a NAALADase inhibitor.

FIG. 30 is a bar graph plotting motor and sensory nerve conductionvelocities of non-diabetic mice and db/db diabetic mice after treatmentwith3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)benzenepropanoicacid (“Compound F”).

FIG. 31 is a 27,000× magnified photograph of a retinal blood vessel froma control, non-diabetic rat.

FIG. 32 is a 27,000× magnified photograph of a retinal blood vessel froma diabetic rat after six months of treatment with a vehicle.

FIG. 33 is a 27,000× magnified photograph of a retinal blood vessel froma diabetic rat after six months of treatment with Compound B.

FIG. 34 is bar graph comparing the rotarod performance of transgenic HDmice and normal non-HD mice treated with Compound B, and transgenic HDmice and normal non-HD mice treated with a vehicle.

FIG. 35 is a bar graph comparing the total distance traveled bytransgenic HD mice and normal non-HD mice treated with Compound B, andtransgenic HD mice and normal non-HD mice treated with a vehicle.

FIG. 36 is a graph plotting the survival time of transgenic HD micetreated with Compound B or a vehicle.

FIG. 37A is a graph plotting the survival time of male transgenic HDmice treated with Compound B or a vehicle.

FIG. 37B is a graph plotting the survival time of female transgenic HDmice treated with Compound B or a vehicle.

DETAILED DESCRIPTION Definitions

“Compound A” refers to2-[[2,3,4,5,6-pentafluorobenzyl)hydroxyphosphinyl]methyl]pentanedioicacid.

“Compound B” refers to 2-(3-sulfanylpropyl)-pentanedioic acid.

“Compound C” refers to 2-(phosphonomethyl)-pentanedioic acid (“PMPA”).

“Compound D” refers to 2-(2-sulfanylethyl)-pentanedioic acid.

“Compound E” refers to3-carboxy-alpha-(3-mercaptopropyl)benzenepropanoic acid.

“Compound F” refers to3-carboxy-5-(1,1-dimethylethyl)-alpha-(3-mercaptopropyl)benzenepropanoicacid.

“Alkyl” refers to a branched or unbranched saturated hydrocarbon chaincomprising a designated number of carbon atoms. For example, C₁–C₉ alkylis a straight or branched hydrocarbon chain containing 1 to 9 carbonatoms, and includes but is not limited to substituents such as methyl,ethyl, propyl, iso-propyl, butyl, iso-butyl, tert-butyl, n-pentyl,n-hexyl, and the like, unless otherwise indicated.

“Alkenyl” refers to a branched or unbranched unsaturated hydrocarbonchain comprising a designated number of carbon atoms. For example, C₂–C₉alkenyl is a straight or branched hydrocarbon chain containing 2 to 9carbon atoms having at least one double bond, and includes but is notlimited to substituents such as ethenyl, propenyl, iso-propenyl,butenyl, iso-butenyl, tert-butenyl, n-pentenyl, n-hexenyl, and the like,unless otherwise indicated.

“Alkoxy” refers to the group —OR wherein R is alkyl as herein defined.In one embodiment, R is a branched or unbranched saturated hydrocarbonchain containing 1 to 9 carbon atoms.

“Carbocycle” refers to a hydrocarbon, cyclic moiety having one or moreclosed ring(s) that is/are alicyclic, aromatic, fused and/or bridged.Examples include cyclopropane, cyclobutane, cyclopentane, cyclohexane,cycloheptane, cyclopentene, cyclohexene, cycloheptene, cyclooctene,benzyl, naphthene, anthracene, phenanthracene, biphenyl and pyrene.

“Aryl” refers to an aromatic, hydrocarbon cyclic moiety having one ormore closed rings. Examples include, without limitation, phenyl, benzyl,naphthyl, anthracenyl, phenanthracenyl, biphenyl and pyrenyl.

“Heterocycle” refers to a cyclic moiety having one or more closed ringsthat is/are alicyclic, aromatic, fused and/or bridged, with one or moreheteroatoms (for example, sulfur, nitrogen or oxygen) in at least one ofthe rings. Examples include, without limitation, pyrrolidine, pyrrole,thiazole, thiophene, piperidine, pyridine, isoxazolidine and isoxazole.

“Heteroaryl” refers to an aromatic, cyclic moiety having one or moreclosed rings with one or more heteroatoms (for example, sulfur, nitrogenor oxygen) in at least one of the rings. Examples include, withoutlimitation, pyrrole, thiophene, pyridine and isoxazole.

“Derivative” refers to a substance produced from another substanceeither directly or by modification or partial substitution.

“Effective amount” refers to the amount required to produce the desiredeffect, for example, to inhibit NAALADase enzyme activity and/orangiogenesis, to effect neuronal activity or TGF-β activity, and/or totreat glutamate abnormality, compulsive disorder, prostate disease,cancer, glaucoma or retinal disorder.

“Electromagnetic radiation” includes without limitation radiation havingthe wavelength of 10⁻²⁰ to 10⁰ meters. Examples include, withoutlimitation, gamma radiation (10⁻²⁰ to 10⁻¹³ m), X-ray radiation (10⁻¹¹to 10⁻⁹ m), ultraviolet light (10 nm to 400 nm), visible light (400 nmto 700 nm), infrared radiation (700 nm to 1.0 mm) and microwaveradiation (1 mm to 30 cm).

“Halo” refers to at least one fluoro, chloro, bromo or iodo moiety.

“Isosteres” refer to elements, functional groups, substituents,molecules or ions having different molecular formulae but exhibitingsimilar or identical physical properties. For example, tetrazole is anisostere of carboxylic acid because it mimics the properties ofcarboxylic acid even though they both have different molecular formulae.Typically, two isosteric molecules have similar or identical volumes andshapes. Ideally, isosteric compounds should be isomorphic and able toco-crystallize. Other physical properties that isosteric compoundsusually share include boiling point, density, viscosity and thermalconductivity. However, certain properties are usually different: dipolarmoments, polarity, polarization, size and shape since the externalorbitals may be hybridized differently. The term “isosteres” encompasses“bioisosteres”.

“Bioisosteres” are isosteres that, in addition to their physicalsimilarities, share some common biological properties. Typically,bioisosteres interact with the same recognition site or produce broadlysimilar biological effects.

“Carboxylic acid isosteres” include without limitation directderivatives such as hydroxamic acids, acylcyanamides andacylsulfonamides; planar acidic heterocycles such as tetrazoles,mercaptoazoles, sulfinylazoles, sulfonylazoles, isoxazoles,isothiazoles, hydroxythiadiazoles and hydroxychromes; and nonplanarsulfur- or phosphorus-derived acidic functions such as phosphinates,phosphonates, phosphonamides, sulphonates, sulphonamides, andacylsulphonamides.

“Metabolite” refers to a substance produced by metabolism or by ametabolic process.

“NAAG” refers to N-acetyl-aspartyl-glutamate, an important peptidecomponent of the brain, with levels comparable to the major inhibitorneurotransmitter gamma-aminobutyric acid (GABA). NAAG isneuron-specific, present in synaptic vesicles and released upon neuronalstimulation in several systems presumed to be glutamatergic. Studiessuggest that NAAG may function as a neurotransmitter and/orneuromodulator in the central nervous system, or as a precursor of theneurotransmitter glutamate. In addition, NAAG is an agonist at group IImetabotropic glutamate receptors, specifically mGluR3 receptors; whenattached to a moiety capable of inhibiting NAALADase, it is expectedthat metabotropic glutamate receptor ligands will provide potent andspecific NAALADase inhibitors.

“NAALADase” refers to N-acetylated α-linked acidic dipeptidase, amembrane bound metallopeptidase which catabolizes NAAG toN-acetylaspartate (“NAA”) and glutamate (“GLU”):

Catabolism of NAAG by NAALADase

NAALADase has been assigned to the M28 peptidase family and is alsocalled PSMA or human GCP II, EC number 3.4.17.21. It is believed thatNAALADase is a co-catalytic zinc/zinc metallopeptidase. NAALADase showsa high affinity for NAAG with a Km of 540 nM. If NAAG is a bioactivepeptide, then NAALADase may serve to inactivate NAAG'S synaptic action.Alternatively, if NAAG functions as a precursor for glutamate, theprimary function of NAALADase may be to regulate synaptic glutamateavailability.

“Pharmaceutically acceptable carrier” refers to any carrier, diluent,excipient, wetting agent, buffering agent, suspending agent, lubricatingagent, adjuvant, vehicle, delivery system, emulsifier, disintegrant,absorbent, preservative, surfactant, colorant, flavorant, or sweetener,preferably non-toxic, that would be suitable for use in a pharmaceuticalcomposition.

“Pharmaceutically acceptable equivalent” includes, without limitation,pharmaceutically acceptable salts, hydrates, metabolites, prodrugs andisosteres. Many pharmaceutically acceptable equivalents are expected tohave the same or similar in vitro or in vivo activity as the inventivecompounds.

“Pharmaceutically acceptable salt” refers to a salt of the inventivecompounds which possesses the desired pharmacological activity and whichis neither biologically nor otherwise undesirable. The salt can beformed with acids that include, without limitation, acetate, adipate,alginate, aspartate, benzoate, benzenesulfonate, bisulfate butyrate,citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloridehydrobromide, hydroiodide, 2-hydroxyethane-sulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,thiocyanate, tosylate and undecanoate. Examples of a base salt includeammonium salts, alkali metal salts such as sodium and potassium salts,alkaline earth metal salts such as calcium and magnesium salts, saltswith organic bases such as dicyclohexylamine salts,N-methyl-D-glucamine, and salts with amino acids such as arginine andlysine. Basic nitrogen-containing groups can be quarternized with agentsincluding lower alkyl halides such as methyl, ethyl, propyl and butylchlorides, bromides and iodides; dialkyl sulfates such as dimethyl,diethyl, dibutyl and diamyl sulfates; long chain halides such as decyl,lauryl, myristyl and stearyl chlorides, bromides and iodides; andaralkyl halides such as benzyl and phenethyl bromides.

“Prodrug” refers to a derivative of the inventive compounds thatundergoes biotransformation, such as metabolism, before exhibiting itspharmacological effect(s). The prodrug is formulated with theobjective(s) of improved chemical stability, improved patient acceptanceand compliance, improved bioavailability, prolonged duration of action,improved organ selectivity, improved formulation (e.g., increasedhydrosolubility), and/or decreased side effects (e.g., toxicity). Theprodrug can be readily prepared from the inventive compounds usingmethods known in the art, such as those described by Burger's MedicinalChemistry and Drug Chemistry, Fifth Ed., Vol. 1, pp. 172–178, 949–982(1995).

“Radiosensitizer” refers to a low molecular weight compound administeredto animals in therapeutically effective amounts to promote the treatmentof diseases that are treatable with electromagnetic radiation. Diseasesthat are treatable with electromagnetic radiation include, withoutlimitation, neoplastic diseases, benign and malignant tumors, andcancerous cells. Electromagnetic radiation treatment of other diseasesnot listed herein are also contemplated by this invention.

“Inhibition,” in the context of enzymes, refers to reversible enzymeinhibition such as competitive, uncompetitive and non-competitiveinhibition. Competitive, uncompetitive and non-competitive inhibitioncan be distinguished by the effects of an inhibitor on the reactionkinetics of an enzyme. Competitive inhibition occurs when the inhibitorcombines reversibly with the enzyme in such a way that it competes witha normal substrate for binding at the active site. The affinity betweenthe inhibitor and the enzyme may be measured by the inhibitor constant,K_(i), which is defined as:

$K_{i} = \frac{\lbrack E\rbrack\lbrack I\rbrack}{\lbrack{EI}\rbrack}$wherein [E] is the concentration of the enzyme, [I] is the concentrationof the inhibitor, and [EI] is the concentration of the enzyme-inhibitorcomplex formed by the reaction of the enzyme with the inhibitor. Unlessotherwise specified, K_(i) as used herein refers to the affinity betweenthe inventive compounds and NAALADase. “IC₅₀” is a related term used todefine the concentration or amount of a compound that is required tocause a 50% inhibition of the target enzyme.

“NAALADase inhibitor” refers to any compound that inhibits NAALADaseenzyme activity. Embodiments include NAALADase inhibitors that exhibit aK_(i) of less than 100 μM, less than 10 μM or less than 1 μM, asdetermined using any appropriate assay known in the art.

“Isomers” refer to compounds having the same number and kind of atoms,and hence the same molecular weight, but differing in respect to thearrangement or configuration of the atoms.

“Optical isomers” refer to enantiomers or diastereoisomers.

“Stereoisomers” are isomers that differ only in the arrangement of theatoms in space.

“Diastereoisomers” are stereoisomers that are not mirror images of eachother. Diastereoisomers occur in compounds having two or more asymmetriccarbon atoms; thus, such compounds have 2^(n) optical isomers, where nis the number of asymmetric carbon atoms.

“Enantiomers” are a pair of stereoisomers that are non-superimposablemirror images of each other. Enantiomers result from the presence of oneor more asymmetric carbon atoms in the compound (e.g., glyceraldehyde,lactic acid, sugars, tartaric acid, amino acids).

“Enantiomer-enriched” refers to a mixture in which one enantiomerpredominates.

“Racemic mixture” means a mixture containing equal amounts of individualenantiomers.

“Non-racemic mixture” is a mixture containing unequal amounts ofenantiomers.

“Angiogenesis” refers to the process whereby new capillaries are formed.“Inhibition” of angiogenesis may be measured by many parameters inaccordance with this invention and, for instance, may be assessed bydelayed appearance of neovascular structures, slowed development ofneovascular structures, decreased occurrence of neovascular structures,slowed or decreased severity of angiogenesis-dependent disease effects,arrested angiogenic growth, or regression of previous angiogenic growth.In the extreme, complete inhibition is referred to herein as prevention.In relation to angiogenesis or angiogenic growth, “prevention” refers tono substantial angiogenesis or angiogenic growth if none had previouslyoccurred, or no substantial further angiogenesis or angiogenic growth ifgrowth had previously occurred.

“Angiogenesis-dependent disease” includes, without limitation,rheumatoid arthritis, cardiovascular diseases, neovascular diseases ofthe eye, peripheral vascular disorders, dermatologic ulcers andcancerous tumor growth, invasion and metastasis.

“Animal” refers to a living organism having sensation and the power ofvoluntary movement, and which requires for its existence oxygen andorganic food. Examples include, without limitation, members of thehuman, equine, porcine, bovine, murine, canine, or feline species. Inthe case of a human, an “animal” may also be referred to as a “patient”.

“Mammal” refers to a warm-blooded vertebrate animal.

“Anxiety” includes without limitation the unpleasant emotion stateconsisting of psychophysiological responses to anticipation of unreal orimagined danger, ostensibly resulting from unrecognized intrapsychicconflict. Physiological concomitants include increased heart rate,altered respiration rate, sweating, trembling, weakness, and fatigue;psychological concomitants include feelings of impending danger,powerlessness, apprehension, and tension. Dorland's Illustrated MedicalDictionary, W.B. Saunders Co., 27th ed. (1988).

“Anxiety Disorder” includes without limitation mental disorders in whichanxiety and avoidance behavior predominate. Dorland's IllustratedMedical Dictionary, W.B. Saunders Co., 27th ed. (1988). Examples includewithout limitation panic attack, agoraphobia, panic disorder, acutestress disorder, chronic stress disorder, specific phobia, simplephobia, social phobia, substance induced anxiety disorder, organicanxiety disorder, obsessive compulsive disorder, post-traumatic stressdisorder, generalized anxiety disorder, and anxiety disorder NOS. Otheranxiety disorders are characterized in Diagnostic and Statistical Manualof Mental Disorders (American Psychiatric Association 4th ed. 1994).

“Attention Deficit Disorder” or “ADD” refers to a disorder characterizedby developmentally inappropriate inattention and impulsiveness, with orwithout hyperactivity. Inattention means a failure to finish tasksstarted, easily distracted, seeming lack of attention, and difficultyconcentrating on tasks requiring sustained attention. Impulsivenessmeans acting before thinking, difficulty taking turns, problemsorganizing work, and constant shifting from one activity to another.Hyperactivity means difficulty staying seated and sitting still, andrunning or climbing excessively.

“Cancer” includes, without limitation, ACTH-producing tumors, acutelymphocytic leukemia, acute nonlymphocytic leukemia, cancer of theadrenal cortex, bladder cancer, brain cancer, breast cancer, cervixcancer, chronic lymphocytic leukemia, chronic myelocytic leukemia,colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer,esophageal cancer, Ewing's sarcoma, gallbladder cancer, hairy cellleukemia, head and neck cancer, Hodgkin's lymphoma, Kaposi's sarcoma,kidney cancer, liver cancer, lung cancer (small and/or non-small cell),malignant peritoneal effusion, malignant pleural effusion, melanoma,mesothelioma, multiple myeloma, neuroblastoma, non-Hodgkin's lymphoma,osteosarcoma, ovary cancer, ovary (germ cell) cancer, pancreatic cancer,penis cancer, prostate cancer, retinoblastoma, skin cancer, soft-tissuesarcoma, squamous cell carcinomas, stomach cancer, testicular cancer,thyroid cancer, trophoblastic neoplasms, cancer of the uterus, vaginalcancer, cancer of the vulva, and Wilm's tumor.

“Compulsive disorder” refers to any disorder characterized byirresistible impulsive behavior. Examples of compulsive disordersinclude without limitation substance dependence, eating disorders,pathological gambling, ADD and Tourette's syndrome.

“Demyelinating disease” refers to any disease involving damage to orremoval of the myelin sheath naturally surrounding nerve tissue, such asthat defined in U.S. Pat. No. 5,859,046 and International PublicationNo. WO 98/03178, herein incorporated by reference. Examples includewithout limitation peripheral demyelinating diseases (such asGuillain-Barré syndrome, peripheral neuropathies and Charcot-Marie Toothdisease) and central demyelinating diseases (such as multiplesclerosis).

“Disease” refers to any deviation from or interruption of the normalstructure or function of any part, organ or system (or combinations) ofthe body that is manifested by a characteristic set of symptoms andsigns and whose etiology, pathology, and prognosis may be known orunknown. Dorland's Illustrated Medical Dictionary, (W.B. Saunders Co.27th ed. 1988).

“Disorder” refers to any derangement or abnormality of function; amorbid physical or mental state. Dorland's Illustrated MedicalDictionary, (W.B. Saunders Co. 27th ed. 1988).

“Eating disorder” refers to compulsive overeating, obesity or severeobesity. Obesity means body weight of 20% over standard height-weighttables. Severe obesity means over 100% overweight.

“Glaucoma” includes without limitation chronic (idiopathic) open-angleglaucomas (e.g., high-pressure, normal-pressure); pupillary blockglaucomas (e.g., acute angle-closure, subacute angle-closure, chronicangle-closure, combined-mechanism); developmental glaucomas (e.g.,congenital (infantile), juvenile, Anxenfeld-Rieger syndrome, Peters'anomaly, Aniridia); glaucomas associated with other ocular disorders(e.g., glaucomas associated with disorders of the corneal endothelium,iris, ciliary body, lens, retina, choroid and vitreous); glaucomasassociated with elevated episcleral venous pressure (e.g., systemicdiseases with associated elevated intraocular pressure and glaucoma,corticosteroid-induced glaucoma); glaucomas associated with inflammationand trauma (e.g., glaucomas associated with keratitis, episcleritis,scleritis, uveitis, ocular trauma and hemorrhage); glaucomas followingintraocular surgery (e.g., ciliary block (malignant) glaucoma, glaucomasin aphakia and pseudophakia, glaucomas associated with corneal surgery,glaucomas associated with vitreoretinal surgery).

“Glutamate abnormality” refers to any disease, disorder, or condition inwhich glutamate is implicated, including pathological conditionsinvolving elevated levels of glutamate. Examples of glutamateabnormalities include, without limitation, compulsive disorder, spinalcord injury, epilepsy, stroke, ischemia, demyelinating disease,Alzheimer's disease, Parkinson's disease, ALS, Huntington's disease(“HD”), schizophrenia, pain, peripheral neuropathy (including but notlimited to diabetic neuropathy), traumatic brain injury, neuronalinsult, inflammatory disease, anxiety, anxiety disorder, memoryimpairment, glaucoma and retinal disorder.

“Ischemia” refers to localized tissue anemia due to obstruction of theinflow of arterial blood. Global ischemia occurs when blood flow ceasesfor a period of time, as may result from cardiac arrest. Focal ischemiaoccurs when a portion of the body, such as the brain, is deprived of itsnormal blood supply, such as may result from thromboembolytic occlusionof a cerebral vessel, traumatic head injury, edema or brain tumor. Evenif transient, both global and focal ischemia can produce widespreadneuronal damage. Although nerve tissue damage occurs over hours or evendays following the onset of ischemia, some permanent nerve tissue damagemay develop in the initial minutes following cessation of blood flow tothe brain. Much of this damage is attributed to glutamate toxicity andsecondary consequences of reperfusion of the tissue, such as the releaseof vasoactive products by damaged endothelium, and the release ofcytotoxic products, such as free radicals and leukotrienes, by thedamaged tissue.

“Memory impairment” refers to a diminished mental registration,retention or recall of past experiences, knowledge, ideas, sensations,thoughts or impressions. Memory impairment may affect short andlong-term information retention, facility with spatial relationships,memory (rehearsal) strategies, and verbal retrieval and production.Common causes of memory impairment are age, severe head trauma, brainanoxia or ischemia, alcoholic-nutritional diseases, drug intoxicationsand neurodegenerative diseases. For example, memory impairment is acommon feature of neurodegenerative diseases such as Alzheimer's diseaseand senile dementia of the Alzheimer type. Memory impairment also occurswith other kinds of dementia such as multi-infarct dementia, a seniledementia caused by cerebrovascular deficiency, and the Lewy-body variantof Alzheimer's disease with or without association with Parkinson'sdisease. Creutzfeldt-Jakob disease is a rare dementia with which memoryimpairment is associated. It is a spongiform encephalopathy caused bythe prion protein; it may be transmitted from other sufferers or mayarise from gene mutations. Loss of memory is also a common feature ofbrain-damaged patients. Brain damage may occur, for example, after aclassical stroke or as a result of an anaesthetic accident, head trauma,hypoglycemia, carbon monoxide poisoning, lithium intoxication, vitamin(B₁, thiamine and B₁₂) deficiency, or excessive alcohol use. Korsakoff'samnesic psychosis is a rare disorder characterized by profound memoryloss and confabulation, whereby the patient invents stories to concealhis or her memory loss. It is frequently associated with excessivealcohol intake. Memory impairment may furthermore be age-associated; theability to recall information such as names, places and words seems todecrease with increasing age. Transient memory loss may also occur inpatients, suffering from a major depressive disorder, afterelectro-convulsive therapy.

“Mental disorder” refers to any clinically significant behavioral orpsychological syndrome characterized by the presence of distressingsymptoms or significant impairment of functioning. Mental disorders areassumed to result from some psychological or organic dysfunction of theindividual; the concept does not include disturbances that areessentially conflicts between the individual and society (socialdeviance).

“Metastasis” refers to “[t]he ability of cells of a cancer todisseminate and form new foci of growth at noncontiguous sites (i.e., toform metastases).” See Hill, R. P, “Metastasis”, The Basic Science ofOncology, Tannock et al., Eds., McGraw-Hill, New York, pp. 178–195(1992), herein incorporated by reference. “The transition from in situtumor growth to metastatic disease is defined by the ability of tumorcells of the primary site to invade local tissues and to cross tissuebarriers . . . To initiate the metastatic process, carcinoma cells mustfirst penetrate the epithelial basement membrane and then invade theinterstitial stroma. For distant metastases, intravasation requirestumor cell invasion of the subendothelial basement membrane that mustalso be negotiated during tumor cell extravasation . . . The developmentof malignancy is also associated with tumor-induced angiogenesis [which]not only allows for expansion of the primary tumors, but also permitseasy access to the vascular compartment due to defects in the basementmembranes of newly formed vessels.” See Aznavoorian et al., Cancer(1993) 71:1368–1383, herein incorporated by reference.

“Nervous insult” refers to any damage to nervous tissue and anydisability or death resulting therefrom. The cause of nervous insult maybe metabolic, toxic, neurotoxic, iatrogenic, thermal or chemical, andincludes without limitation ischemia, hypoxia, cerebrovascular accident,trauma, surgery, pressure, mass effect, hemorrhage, radiation,vasospasm, neurodegenerative disease, neurodegenerative process,infection, Parkinson's disease, ALS, myelination/demyelinationprocesses, epilepsy, cognitive disorder, glutamate abnormality andsecondary effects thereof.

“Nervous tissue” refers to the various components that make up thenervous system, including without limitation neurons, neural supportcells, glia, Schwann cells, vasculature contained within and supplyingthese structures, the central nervous system, the brain, the brain stem,the spinal cord, the junction of the central nervous system with theperipheral nervous system, the peripheral nervous system and alliedstructures.

“Neuropathy” refers to any disease or malfunction of the nerves.Neuropathy includes, without limitation, peripheral neuropathy, diabeticneuropathy, autonomic neuropathy and mononeuropathy. Peripheralneuropathy may be idiopathic or induced by any causes including diseases(for example, amyloidosis, alcoholism, HIV, syphilis, virus, autoimmunedisorder, cancer, porphyria, arachnoiditis, post herpetic neuralgia,Guillain-Barré syndrome, diabetes including type I and type IIdiabetes), chemicals (for example, toxins, lead, dapsone, vitamins,paclitaxel chemotherapy, HAART therapy) and physical injuries to aparticular nerve or nerve plexus (for example, trauma, compression,constriction).

“Neuroprotective” refers to the effect of reducing, arresting orameliorating nervous insult, and protecting, resuscitating or revivingnervous tissue that has suffered nervous insult.

“Pain” refers to localized sensations of discomfort, distress or agony,resulting from the stimulation of specialized nerve endings. It servesas a protective mechanism insofar as it induces the sufferer to removeor withdraw from the source. Dorland's Illustrated Medical Dictionary,(W.B. Saunders Co. 27th ed. 1988). Examples of pain include, withoutlimitation, acute, chronic, cancer, burn, incisional, inflammatory,neuropathic and back pain.

“Neuropathic pain” refers to a condition of pain associated with a nerveinjury. Depending on the particular syndrome, the pain may be due toalterations of the brain or spinal cord or may be due to abnormalitiesin the nerve itself. Neuropathic pain may be idiopathic or induced byany causes including diseases (for example, amyloidosis, alcoholism,HIV, syphilis, virus, autoimmune disorder, cancer, porphyria,arachnoiditis, post herpetic neuralgia, Guillain-Barré syndrome,diabetes including type I and type II diabetes), chemicals (for example,toxins, lead, dapsone, vitamins, paclitaxel chemotherapy, HAART therapy)and physical injuries to a particular nerve or nerve plexus (forexample, trauma, compression, constriction).

“Pathological gambling” refers to a condition characterized by apreoccupation with gambling. Similar to psychoactive substance abuse,its effects include development of tolerance with a need to gambleprogressively larger amounts of money, withdrawal symptoms, andcontinued gambling despite severe negative effects on family andoccupation.

“Prostate disease” refers to any disease affecting the prostate.Examples of prostate disease include without limitation prostate cancersuch as adenocarcinoma and metastatic cancers of the prostate; andconditions characterized by abnormal growth of prostatic epithelialcells such as benign prostatic hyperplasia.

“Retinal disorder” refers to vascular retinopathy, for example,hypertensive retinopathy, diabetic retinopathy (nonproliferative orproliferative), central retinal artery occlusion, or central retinalvein occlusion; age-related macular degeneration; retinal detachment; orretinitis pigmentosa.

“Schizophrenia” refers to a mental disorder or group of mental disorderscharacterized by disturbances in form and content of thought (looseningof associations, delusions, hallucinations), mood (blunted, flattened,inappropriate affect), sense of self and relationship to the externalworld (loss of ego boundaries, dereistic thinking, and autisticwithdrawal), and behavior (bizarre, apparently purposeless, andstereotyped activity or inactivity). Examples of schizophrenia include,without limitation, acute, ambulatory, borderline, catatonic, childhood,disorganized, hebephrenic, latent, nuclear, paranoid, paraphrenic,prepsychotic, process, pseudoneurotic, pseudopsychopathic, reactive,residual, schizo-affective and undifferentiated schizophrenia. Dorland'sIllustrated Medical Dictionary, (W.B. Saunders Co. 27th ed. 1988).

“TGF-β” refers to transforming growth factor beta. TGF-β is recognizedas a prototype of multifunctional growth factors. It regulates variouscell and tissue functions, including cell growth and differentiation,angiogenesis, wound healing, immune function, extracellular matrixproduction, cell chemotaxis, apoptosis and hematopoiesis.

“TGF-β abnormality” refers to any disease, disorder or condition inwhich TGF-β is implicated, including diseases disorders and conditionscharacterized by an abnormal level of TGF-β.

“Abnormal level of TGF-β” refers to a measurable variance from normallevels of TGF-β, as determined by one of ordinary skill in the art usingknown techniques.

“Therapeutic window of opportunity” or “window” refers, in relation tostroke, to the maximal delay between the onset of stroke and theinitiation of efficacious therapy.

“Tourette's syndrome” refers to an autosomal multiple tic disordercharacterized by compulsive swearing, multiple muscle tics and loudnoises. Tics are brief, rapid, involuntary movements that can be simpleor complex; they are stereotyped and repetitive, but not rhythmic.Simple tics, such as eye blinking, often begin as nervous mannerisms.Complex tics often resemble fragments of normal behavior.

Unless otherwise defined in conjunction with specific diseases ordisorders, “treating” refers to:

(i) preventing a disease, disorder or condition from occurring in ananimal that may be predisposed to the disease, disorder and/or conditionbut has not yet been diagnosed as having it;

(ii) inhibiting the disease, disorder or condition, i.e., arresting itsdevelopment; and/or

(iii) relieving the disease, disorder or condition, i.e., causingregression of the disease, disorder and/or condition.

“Treating ALS” refers to:

(i) preventing ALS from occurring in an animal that may be predisposedto ALS but has not yet been diagnosed as having it;

(ii) inhibiting ALS, e.g. arresting its development;

(iii) relieving ALS, e.g. causing regression of the disease, disorderand/or condition;

(iv) delaying onset of ALS or ALS symptom(s);

(v) slowing progression of ALS or ALS symptom(s);

(vi) prolonging survival of an animal suffering from ALS; and/or

(vii) attenuating ALS symptom(s).

“Treating substance dependence” refers to preventing relapse; reducingcraving; suppressing tolerance; preventing, inhibiting and/or relievingwithdrawal; attenuating sensitization; preventing, inhibiting (i.e.arresting development of) and/or relieving (i.e. causing regression of)substance-induced neurotoxicity; and/or preventing, inhibiting and/orrelieving fetal alcohol syndrome.

“Craving” refers to a strong desire for a substance and/or a compellingurge and/or an irresistible impulse to use a substance.

“Dependence” refers to a maladaptive pattern of substance use, leadingto clinically significant impairment or distress. Dependence istypically characterized by tolerance and/or withdrawal. Substances forwhich dependence may be developed include, without limitation,depressants (opioids, synthetic narcotics, barbiturates, glutethimide,methyprylon, ethchlorvynol, methaqualone, alcohol); anxiolytics(diazepam, chlordiazepoxide, alprazolam, oxazepam, temazepam);stimulants (amphetamine, methamphetamine, cocaine); and hallucinogens(LSD, mescaline, peyote, marijuana).

“Relapse” refers to a return to substance use after a period ofabstinence, often accompanied by reinstatement.

“Reinstatement” refers to a return to a preexisting level of use anddependence in a person who has resumed substance use following a periodof abstinence.

“Sensitization” refers to a condition in which the response to asubstance increases with repeated use.

“Tolerance” refers to an acquired reaction to a substance characterizedby diminished effect with continued use of the same dose and/or a needfor increased doses to achieve intoxication or desired effect previouslyachieved by lower doses. Both physiological and psychosocial factors maycontribute to the development of tolerance. With respect tophysiological tolerance, metabolic and/or functional tolerance maydevelop. By increasing the rate of metabolism of the substance, the bodymay be able to eliminate the substance more readily. Functionaltolerance is defined as a decrease in sensitivity of the central nervoussystem to the substance.

“Withdrawal” refers to a syndrome characterized by untoward physicalchanges that occur following cessation of or reduction in substance use,or administration of a pharmacologic antagonist.

One of ordinary skill in the art will recognize that there arealternative nomenclatures, nosologies and classification systems for thediseases, disorders and conditions defined above, and that such systemsevolve with medical scientific progress.

Unless the context clearly dictates otherwise, the definitions ofsingular terms may be extrapolated to apply to their plural counterpartsas they appear in the application; likewise, the definitions of pluralterms may be extrapolated to apply to their singular counterparts asthey appear in the application.

Compounds of the Invention

This invention relates to a compound of formula I

or a pharmaceutically acceptable equivalent of said compound, wherein:

A¹, A², A³ and A⁴ are independently hydrogen, C₁–C₉ alkyl, C₂–C₉alkenyl, C₂–C₉ alkynyl, aryl, heteroaryl, carbocycle, heterocycle, C₁–C₉alkoxy, C₂–C₉ alkenyloxy, phenoxy, benzyloxy, hydroxy, halo, nitro,cyano, isocyano, —COOR⁶, —COR⁶, —NR⁶R⁷, —SR⁶, —SOR⁶, —SO₂R⁶, —SO₂(OR⁶),—(C═O)NR⁶R⁷, —(C═O)NR⁶(CH₂)_(n)COOH, —NR⁶(C═O)R⁷ or —(CH₂)_(n)COOH, orany adjacent two of A¹, A², A³ and A⁴ form with the benzene ring a fusedring that is saturated or unsaturated, aromatic or non-aromatic, andcarbocyclic or heterocyclic, said heterocyclic ring containing 1 or 2oxygen, nitrogen and/or sulfur heteroatom(s);

n is 1–3;

R, R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ are independently hydrogen, carboxy,C₁–C₉ alkyl, C₂–C₉ alkenyl, C₂–C₉ alkynyl, aryl, heteroaryl, carbocycleor heterocycle; and

said alkyl, alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle,alkoxy, alkenyloxy, phenoxy, benzyloxy and fused ring are independentlyunsubstituted or substituted with one or more substituent(s).

In one embodiment, A¹, A², A³ and A⁴ are independently hydrogen and—COOH; R¹, R², R³ and R⁴ are each hydrogen; and R⁵ is hydrogen, phenyl,benzyl or phenylethyl, wherein said phenyl, benzyl and phenylethyl areindependently unsubstituted or substituted with one or moresubstituent(s). In another embodiment, R⁵ is benzyl substituted with oneor more substituent(s) independently selected from the group consistingof carboxy, halo, C₁–C₄ alkyl and C₁–C₄ alkoxy.

Possible substituents of said alkyl, alkenyl, alkynyl, aryl, heteroaryl,carbocycle, heterocycle, alkoxy, alkenyloxy, phenoxy, benzyloxy andfused ring include, without limitation, C₁–C₆ alkyl, C₂–C₆ alkenyl,C₂–C₆ alkynyl, C₁–C₆ alkoxy, C₂–C₆ alkenyloxy, phenoxy, benzyloxy,hydroxy, carboxy, hydroperoxy, carbamido, carbamoyl, carbamyl, carbonyl,carbozoyl, amino, hydroxyamino, formamido, formyl, guanyl, cyano,cyanoamino, isocyano, isocyanato, diazo, azido, hydrazino, triazano,nitrilo, nitro, nitroso, isonitroso, nitrosamino, imino, nitrosimino,oxo, C₁–C₆ alkylthio, sulfamino, sulfamoyl, sulfeno, sulfhydryl,sulfinyl, sulfo, sulfonyl, thiocarboxy, thiocyano, isothiocyano,thioformamido, halo, haloalkyl, chlorosyl, chloryl, perchloryl,trifluoromethyl, iodosyl, iodyl, phosphino, phosphinyl, phospho,phosphono, arsino, selanyl, disilanyl, siloxy, silyl, silylene andcarbocyclic and heterocyclic moieties. Carbocyclic moieties includealicyclic and aromatic structures.

Examples of carbocyclic and heterocyclic moieties include, withoutlimitation, phenyl, benzyl, naphthyl, indenyl, azulenyl, fluorenyl,anthracenyl, indolyl, isoindolyl, indolinyl, benzofuranyl,benzothiophenyl, indazolyl, benzimidazolyl, benzthiazolyl,tetrahydrofuranyl, tetrahydropyranyl, pyridyl, pyrrolyl, pyrrolidinyl,pyridinyl, pyrimidinyl, purinyl, quinolinyl, isoquinolinyl,tetrahydroquinolinyl, quinolizinyl, furyl, thiophenyl, imidazolyl,oxazolyl, benzoxazolyl, thiazolyl, isoxazolyl, isotriazolyl,oxadiazolyl, triazolyl, thiadiazolyl, pyridazinyl, pyrimidinyl,pyrazinyl, triazinyl, trithianyl, indolizinyl, pyrazolyl, pyrazolinyl,pyrazolidinyl, thienyl, tetrahydroisoquinolinyl, cinnolinyl,phthalazinyl, quinazolinyl, quinoxalinyl, naphthyridinyl, pteridinyl,carbazolyl, acridinyl, phenazinyl, phenothiazinyl, and phenoxazinyl.

Representative compounds of the invention are set forth below in TABLEI.

TABLE I Compound No. Structure/Name 1

3-(2-mercaptoethyl)-1H-indole-2- carboxylic acid 2

3-(2-mercaptoethyl)-1H-indole-2,7- dicarboxylic acid 3

1-[(3-carboxyphenyl)methyl]-3-(2- mercaptoethyl)-1H-indole-2-carboxylicacid 4

1-[(2-bromo-5-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid 5

1-[(4-carboxyphenyl)methyl]-3-(2- mercaptoethyl)-1H-indole-2-carboxylicacid 6

3-(2-mercaptoethyl)-1-(phenylmethyl)-1H- indole-2-carboxylic acid 7

1-[(2-carboxyphenyl)methyl]-3-(2- mercaptoethyl)-1H-indole-2-carboxylicacid 8

1-[[3-carboxy-5-(1,1-dimethylethyl)-phenyl]methyl]-3-(2-mercaptoethyl)-1H- indole-2-carboxylic acid 9

1-[(4-bromo-3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid 10

1-[(2-carboxy-5-methoxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid 11

3-(2-mercaptoethyl)-1-phenyl-1H-indole-2- carboxylic acid 12

3-(2-mercaptoethyl)-1-(2-phenylethyl)-1H- indole-2-carboxylic acid 13

1-(3-carboxyphenyl)-3-(2-mercaptoethyl)- 1H-indole-2-carboxylic acid 14

1-[3-carboxy-5-(1,1-dimethylethyl)-phenyl]-3-(2-mercaptoethyl)-1H-indole-2- carboxylic acid

The inventive compounds may possess one or more asymmetric carboncenter(s) and, thus, may be capable of existing in the form of opticalisomers as well as in the form of racemic or non-racemic mixtures ofoptical isomers. The optical isomers can be obtained by resolution ofthe racemic mixtures according to conventional processes well known inthe art, for example by formation of diastereoisomeric salts bytreatment with an optically active acid or base and then separation ofthe mixture of diastereoisomers by crystallization followed byliberation of the optically active bases from these salts. Examples ofuseful acids include tartaric, diacetyltartaric, dibenzoyltartaric,ditoluoyltartaric and camphorsulfonic acids.

A different process for separating optical isomers involves the use of achiral chromatography column optimally chosen to maximize the separationof the enantiomers. Still another available method involves synthesis ofcovalent diastereoisomeric molecules, for example, esters, amides,acetals, ketals, and the like, by reacting compounds used in theinventive methods and pharmaceutical compositions with an opticallyactive acid in an activated form, an optically active diol or anoptically active isocyanate. The synthesized diastereoisomers can beseparated by conventional means, such as chromatography, distillation,crystallization or sublimation, and then hydrolyzed to deliver theenantiomerically pure compound. In some cases, hydrolysis to the parentoptically active drug prior to dosing the patient is unnecessary sincethe compound can behave as a prodrug. The optically active compounds ofthe invention can likewise be obtained by utilizing optically activestarting materials.

It is understood that the inventive compounds encompass optical isomersas well as racemic and non-racemic mixtures.

Methods of the Invention Methods for Inhibiting NAALADase EnzymeActivity

This invention relates to a method for inhibiting NAALADase enzyme,comprising administering to an animal or mammal in need of suchinhibition an effective amount of a compound of the invention, asdefined above.

Methods for Treating Glutamate Abnormalities

This invention further relates to a method for treating a glutamateabnormality, comprising administering to an animal or mammal in need ofsuch treatment an effective amount of a compound of the invention, asdefined above.

Glutamate abnormalities to be treated include compulsive disorder,stroke, ischemia, demyelinating disease, Parkinson's disease, ALS, HD,schizophrenia, pain, anxiety, anxiety disorder, memory impairment,glaucoma and retinal disorder. Compulsive disorder may be, but is notlimited to, alcohol, nicotine or cocaine dependence.

Stroke patients often experience a significant temporal delay betweenthe onset of ischemia and the initiation of therapy. Thus, there is aneed for neuroprotectants with a long therapeutic window of opportunity.It is expected that the inventive compounds have a therapeutic window ofopportunity of at least 1 hour. Accordingly, when the glutamateabnormality is stroke, the compound of the invention may be administeredto said animal or mammal for up to 60 minutes, 120 minutes or morefollowing onset of stroke.

Without being bound to any particular mechanism of action, compounds ofthe invention are expected to include those that block glutamate releasepre-synaptically without interacting with post-synaptic glutamatereceptors. Such compounds would be devoid of the behavioral toxicitiesassociated with post-synaptic glutamate antagonists.

Methods for Effecting Neuronal Activities

This invention further relates to a method for effecting a neuronalactivity, comprising administering to an animal or mammal in need ofsuch effect an effective amount of a compound of the invention, asdefined above.

The neuronal activity that is effected by the inventive method may bestimulation of damaged neurons, promotion of neuronal regeneration,prevention of neurodegeneration or treatment of a neurological disorder.

Examples of neurological disorders that are treatable by the methods ofthis invention include without limitation: trigeminal neuralgia;glossopharyngeal neuralgia; Bell's Palsy; myasthenia gravis; musculardystrophy; ALS; progressive muscular atrophy; progressive bulbarinherited muscular atrophy; herniated, ruptured or prolapsedinvertebrate disk syndromes; cervical spondylosis; plexus disorders;thoracic outlet destruction syndromes; neuropathy; pain; Alzheimer'sdisease; Parkinson's disease; ALS; and HD.

The inventive method is particularly useful for treating a neurologicaldisorder selected from the group consisting of neuropathy (includingperipheral neuropathy and diabetic neuropathy), pain (includingneuropathic pain such as neuropathic pain induced by diabetes),traumatic brain injury, physical damage to spinal cord, strokeassociated with brain damage, demyelinating disease and neurologicaldisorder relating to neurodegeneration.

When the neurological disorder is pain, the compound of the inventionmay be administered in combination with an effective amount of morphine.

Examples of neurological disorders relating to neurodegeneration includeAlzheimer's disease, Parkinson's disease, ALS and HD.

Methods for Treating Prostate Diseases

This invention further relates to a method for treating a prostatedisease, comprising administering to an animal or mammal in need of suchtreatment an effective amount of a compound of the invention, as definedabove.

Methods for Treating Cancers

This invention further relates to a method for treating cancer,comprising administering to an animal or mammal in need of suchtreatment an effective amount of a compound of the invention, as definedabove.

Cancers to be treated include those in tissues where NAALADase resides,including without limitation the brain, kidney and testis.

Methods for Inhibiting Angiogenesis

This invention further relates to a method for inhibiting angiogenesis,comprising administering to an animal or mammal in need of suchinhibition an effective amount of a compound of the invention, asdefined above.

Angiogenesis may be necessary for fertility or metastasis of cancertumors, or may be related to an angiogenic-dependent disease. Thus, theinventive methods may also be useful for treating anangiogenic-dependent disease including, without limitation, rheumatoidarthritis, cardiovascular diseases, neovascular diseases of the eye,peripheral vascular disorders, dermatologic ulcers and cancerous tumorgrowth, invasion or metastasis.

Methods for Effecting TGF-β Activity

This invention further relates to a method for effecting a TGF-βactivity, comprising administering to an animal or mammal in need ofsuch effect an effective amount of a compound of the invention, asdefined above.

Said effecting a TGF-β activity includes increasing, reducing orregulating TGF-β levels, and treating TGF-β abnormalities. Examples ofTGF-β abnormalities to be treated include neurodegenerative disorders,extra-cellular matrix formation disorders, cell-growth related diseases,infectious diseases, immune related diseases, epithelial tissuescarring, collagen vascular diseases, fibroproliferative disorders,connective tissue disorders, inflammation, inflammatory diseases,respiratory distress syndrome, infertility and diabetes.

Typical neurodegenerative disorders to be treated include neural tissuedamage resulting from ischemia reperfusion injury, myelination andneurodegeneration.

Typical cell-growth related disorders to be treated include thoseaffecting kidney cells, hematopoietic cells, lymphocytes, epithelialcells and endothelial cells.

Typical infectious diseases to be treated include those caused by amacrophage pathogen, particularly a macrophage pathogen selected fromthe group consisting of bacteria, yeast, fungi, viruses, protozoa,Trypanosoma cruzi, Histoplasma capsulatum, Candida albicans, Candidaparapsilosis, Cryptococcus neoformans, Salmonella, Pneumocystis,Toxoplasma, Listeria, Mycobacteria, Rickettsia and Leishmania.Mycobacteria include without limitation Mycobacterium tuberculosis andMycobacterium leprae. Toxoplasma includes without limitation Toxoplasmagondii. Rickettsia includes without limitation R. prowazekii, R. coroniiand R. tsutsugamushi.

Other examples of infectious diseases to be treated include single ormultiple cutaneous lesions, mucosal disease, Chagas' disease, acquiredimmunodeficiency syndrome (AIDS), toxoplasmosis, leishmaniasis,trypanosomiasis, shistosomiasis, cryptosporidiosis, Mycobacterium aviuminfections, Pneumocystis carinii pneumonia and leprosy.

Typical immune related diseases to be treated include autoimmunedisorders; impaired immune function; and immunosuppression associatedwith an infectious disease, particularly, trypanosomal infection, viralinfection, human immunosuppression virus, human T cell lymphotropicvirus (HTLV-1), lymphocytic choriomeningitis virus or hepatitis.

Typical collagen vascular diseases to be treated include progressivesystemic sclerosis (

PSS

), polymyositis, scleroderma, dermatomyositis, eosinophilic fascitis,morphea, Raynaud's syndrome, interstitial pulmonary fibrosis,scleroderma and systemic lupus erythematosus.

Typical fibroproliferative disorders to be treated include diabeticnephropathy, kidney disease, proliferative vitreoretinopathy, livercirrhosis, biliary fibrosis, and myelofibrosis. Kidney diseases include,but are not limited to, mesangial proliferative glomerulonephritis,crescentic glomerulonephritis, diabetic neuropathy, renal interstitialfibrosis, renal fibrosis in transplant patients receiving cyclosporin,and HIV-associated nephropathy.

Typical connective tissue disorders to be treated include scleroderma,myelofibrosis, and hepatic, intraocular and pulmonary fibrosis.

Typical inflammatory diseases to be treated are associated with PSS,polymyositis, scleroderma, dermatomyositis, eosinophilic fascitis,morphea, Raynaud's syndrome, interstitial pulmonary fibrosis,scleroderma, systemic lupus erythematosus, diabetic nephropathy, kidneydisease, proliferative vitreoretinopathy, liver cirrhosis, biliaryfibrosis, myelofibrosis, mesangial proliferative glomerulonephritis,crescentic glomerulonephritis, diabetic neuropathy, renal interstitialfibrosis, renal fibrosis in transplant patients receiving cyclosporin,or HIV-associated nephropathy.

Without being limited to any particular mechanism of action, compoundsof the invention include those that treat inflammatory diseases byregulating TGF-β and/or inhibiting myeloperoxidase.

Other uses associated with the inventive compounds' TGF-β regulatingproperties include:

stimulating growth of tissue, glands or organs, particularly growth thatwould enhance milk production or weight gain;

stimulating cell proliferation, particularly proliferation offibroblasts, mesenchymal cells or epithelial cells;

inhibiting cell growth, particularly of epithelial cells, endothelialcells, T and B lymphocytes and thymocytes;

inhibiting expression of adipose, skeletal muscle and hematopoieticphenotypes, neoplasms, non-cytocidal viral or other pathogenicinfections and autoimmune disorders;

mediating disease resistance and susceptibility;

suppressing cellular immune response;

inhibiting scar tissue formation, such as in skin or other epithelialtissue that has been damaged by wounds resulting from accidental injury,surgical operations, trauma-induced lacerations or other trauma, orwounds involving the peritoneum for which the excessive connectivetissue formation is abdominal adhesions;

increasing the effectiveness of a vaccine, particularly a vaccine for anallergy towards, for example, dust or hayfever; and

inhibiting polyp formation.

Diagnostic Methods and Kits

The inventive compounds are useful for in vitro and in vivo diagnosticmethods for detecting diseases, disorders and conditions where NAALADaselevels are altered including, without limitation, neurologicaldisorders, glutamate abnormalities, neuropathy, pain, compulsivedisorders, prostate diseases, cancers and TGF-β abnormalities.

Accordingly, this invention also relates to a method for detecting adisease, disorder or condition where NAALADase levels are altered,comprising:

(i) contacting a sample of bodily tissue or fluid with a compound of theinvention, as defined above, wherein said compound binds to anyNAALADase in said sample; and

(ii) measuring the amount of any NAALADase bound to said sample, whereinthe amount of NAALADase is diagnostic for said disease, disorder orcondition.

Examples of bodily tissues and fluids include, without limitation,prostate tissue, ejaculate, seminal vesicle fluid, prostatic fluid,urine, blood, saliva, tears, sweat, lymph and sputum.

The compound may be labeled with a marker using techniques known in theart. Useful markers include, without limitation, enzymatic markers andimaging reagents. Examples of imaging reagents include radiolabels suchas ¹³¹I, ¹¹¹In, ¹²³I, ⁹⁹Tc, ³²P, ¹²⁵I, ³H and ¹⁴C; fluorescent labelssuch as fluorescein and rhodamine; and chemiluminescers such asluciferin.

The amount of NAALADase can be measured using techniques known in theart including, without limitation, assays (such as immunometric,calorimetric, densitometric, spectrographic and chromatographic assays)and imaging techniques (such as magnetic resonance spectroscopy (MRS),magnetic resonance imaging (MRI), single-photon emission computedtomography (SPECT) and positron emission tomography (PET)).

This invention further relates to a diagnostic kit for detecting adisease, disorder or condition where NAALADase levels are altered,comprising a compound of the invention, as defined above, labeled with amarker. The kit may further comprise buffering agents, agents forreducing background interference, control reagents and/or apparatus forconducting the test.

This invention further relates to a method for detecting a disease,disorder or condition where NAALADase levels are altered in an animal ora mammal, comprising:

(i) labeling a compound of the invention, as defined above, with animaging reagent;

(ii) administering to said animal or mammal an effective amount of thelabeled compound;

(iii) allowing said labeled compound to localize and bind to NAALADasepresent in said animal or mammal; and

(iv) measuring the amount of NAALADase bound to said labeled compound,wherein the amount of NAALADase is diagnostic for said disease, disorderor condition.

The amount of NAALADase can be measured in vivo using known imagingtechniques, as described above.

Incorporation by Reference

The relationship between NAALADase inhibitors and glutamate, and theeffectiveness of NAALADase inhibitors in treating and detecting variousdiseases, disorders and conditions have been discussed in U.S. Pat. Nos.5,672,592, 5,795,877, 5,804,602, 5,824,662, 5,863,536, 5,977,090,5,981,209, 6,011,021, 6,017,903, 6,025,344, 6,025,345, 6,046,180,6,228,888 and 6,265,609; International Publications Nos. WO 00/01668 andWO 00/38785; and other references generally known in the art. Thepresent inventors hereby incorporate by reference, as though set forthherein in full, the entire contents of the aforementioned patents andpublications, particularly their discussions, figures and data regardingthe effectiveness of NAALADase inhibitors in inhibiting angiogenesis, ineffecting TGF-β activity, in diagnosing diseases, and in treatingischemia, spinal cord injury, demyelinating diseases, Parkinson'sdisease, ALS, alcohol dependence, nicotine dependence, cocainedependence, prostate disease, cancer, diabetic neuropathy, pain,schizophrenia, anxiety, anxiety disorder and memory impairment. Thepresent inventors have discovered that the inventive compounds areeffective NAALADase inhibitors. Thus, the inventive compounds areexpected to have the same uses as the NAALADase inhibitors disclosed inthe patents and publications incorporated by reference.

Pharmaceutical Compositions of the Invention

This invention also relates to a pharmaceutical composition comprising:

(i) an effective amount of a compound of the invention, as definedabove; and

(ii) a pharmaceutically acceptable carrier.

In embodiments, the pharmaceutical composition of the invention ispresent in an effective amount for inhibiting NAALADase enzyme activityor angiogenesis, effecting a neuronal activity or TGF-β activity, ortreating a glutamate abnormality, compulsive disorder, prostate disease,cancer, glaucoma or retinal disorder in an animal or a mammal.

Route of Administration

The inventive compounds and compositions may be administered locally orsystemically by any means known to an ordinarily skilled artisan. Forexample, the inventive compounds and compositions may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir in dosage formulationscontaining conventional non-toxic pharmaceutically acceptable carriers,adjuvants and vehicles. The term parenteral as used herein includessubcutaneous, intravenous, intraarterial, intramuscular,intraperitoneal, intrathecal, intraventricular, intrasternal,intracranial or intraosseous injection and infusion techniques. Theexact administration protocol will vary depending upon various factorsincluding the age, body weight, general health, sex and diet of thepatient; the determination of specific administration procedures wouldbe routine to an ordinarily skilled artisan.

To be effective therapeutically as central nervous system targets, theinventive compounds and compositions should readily penetrate theblood-brain barrier when peripherally administered. Compounds andcompositions that cannot penetrate the blood-brain barrier can beeffectively administered by an intraventricular route or by othermethods recognized in the art. See, for example, U.S. Pat. Nos.5,846,565, 5,651,986 and 5,626,862.

Dosage

The inventive compounds and compositions may be administered by a singledose, multiple discrete doses or continuous infusion. Pump means,particularly subcutaneous pump means, are preferred for continuousinfusion.

Dose levels on the order of about 0.001 to about 10,000 mg/kg of theactive ingredient compound are useful in the treatment of the aboveconditions, with levels including those being about 0.1 to about 1,000mg/kg and about 1 to about 100 mg/kg. The specific dose level for anyparticular patient will vary depending upon a variety of factors,including the activity and the possible toxicity of the specificcompound employed; the age, body weight, general health, sex and diet ofthe patient; the time of administration; the rate of excretion; drugcombination; the severity of the particular disease being treated; andthe form of administration. Typically, in vitro dosage-effect resultsprovide useful guidance on the proper doses for patient administration.Studies in animal models are also helpful. The considerations fordetermining the proper dose levels are well known in the art.

Administration Regimen

Any administration regimen well known to an ordinarily skilled artisanfor regulating the timing and sequence of drug delivery can be used andrepeated as necessary to effect treatment. Such regimen may includepretreatment and/or co-administration with additional therapeuticagents.

Co-Administration with Other Treatments

The inventive compounds and compositions may be used alone or incombination with one or more additional agent(s) for simultaneous,separate or sequential use.

The additional agent(s) may be any therapeutic agent(s) known to anordinarily skilled artisan, including without limitation: one or morecompound(s) of the invention; steroids, for example, hydrocortisonessuch as methylprednisolone; anti-inflammatory or anti-immune drugs, suchas methotrexate, azathioprine, cyclophosphamide or cyclosporin A;interferon-β; antibodies, such as anti-CD4 antibodies; agents which canreduce the risk of a second ischemic event, such as ticlopidine;chemotherapeutic agents; immunotherapeutic compositions; electromagneticradiosensitizers; and morphine.

The inventive compounds and compositions can be co-administered with oneor more therapeutic agents either (i) together in a single formulation,or (ii) separately in individual formulations designed for optimalrelease rates of their respective active agent. Each formulation maycontain from about 0.01% to about 99.99% by weight, including from about3.5% to about 60% by weight, of a compound of this invention, as well asone or more pharmaceutically acceptable carriers.

Preparation of Compounds

The inventive compounds can be readily prepared by standard techniquesof organic chemistry, utilizing the general synthetic pathways depictedbelow in SCHEMES I–IV.

EXAMPLES

The following examples are illustrative of this invention and are notintended to be limitations thereon. Unless otherwise indicated, allpercentages are based upon 100% by weight of the final composition.

Example 1 Preparation of 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid(SCHEME I) 3-(2-Hydroxyethyl)-1H-indole-2-carboxylic acid

To a solution of 4,9-dihydro-3H-pyrano[3,4-b]indol-1-one (Arch. Pharm.,1987, 320, 1202–1209, 5.225 g, 27.9 mmol) in THF (25 mL) was addeddeoxygenated 3 N KOH (25 mL). The mixture was stirred at roomtemperature for 4.5 hours. The reaction mixture was washed with EtOAc,acidified with 1 N HCl, and extracted with EtOAc. The organic extractwas dried over MgSO₄ and concentrated to give 5.589 g of3-(2-hydroxyethyl)-1H-indole-2-carboxylic acid as a solid. This materialwas used for the next reaction without further purification: ¹H NMR(DMSO-d₆) δ 3.26 (t, J=7.5 Hz, 2H), 3.63 (t, J=7.5 Hz, 2H), 7.08 (t,J=7.5 Hz, 1H), 7.26 (t, J=7.7 Hz, 1H), 7.43 (d, J=8.3 Hz, 1H), 7.69 (d,J=8.1 Hz, 1H). Anal. Calcd. for C₁₁H₁₁NO₃.0.1 EtOAc: C., 63.98; H, 5.56;N, 6.54. Found: C, 63.90; H, 5.57; N, 6.46.

Methyl 3-(2-Hydroxyethyl)-1H-indole-2-carboxylate

To a solution of 3-(2-hydroxyethyl)-1H-indole-2-carboxylic acid (5.589g, 26.1 mmol) in methanol (75 mL) was added concentrated H₂SO₄ (3 mL),and the mixture was refluxed overnight. The solvent was removed under areduced pressure and the residual oil was dissolved in EtOAc. Theorganic solution was washed with a saturated aqueous NaHCO₃ solution,dried over Na₂SO₄, and concentrated to give 5.670 g of methyl3-(2-hydroxyethyl)-1H-indole-2-carboxylate as an oil. This material wasused for the next reaction without further purification: ¹H NMR (CDCl₃)δ 2.21 (brs, 1H), 3.40 (t, J=6.5 Hz, 2H), 3.88 (s, 3H), 3.94 (t, J=6.5Hz, 2H), 7.14 (ddd, J=1.7, 6.3, and 8.1 Hz, 1H), 7.2–7.4 (m, 2H), 7.70(d, J=8.1 Hz, 1H), 9.07 (brs, 1H).

Methyl 3-[2-(acetylthio)ethyl]-1H-indole-2-carboxylate

To a solution of triphenylphosphine (1.196 g, 4.56 mmol) in THF (10 mL)was added diisopropyl azodicarboxylate (0.9 mL; 4.56 mmol) at 0° C., andthe mixture was stirred at 0° C. for 30 minutes. A solution of methyl3-(2-hydroxyethyl)-1H-indole-2-carboxylate (1.000 g of the abovematerial) and thioacetic acid (0.34 mL, 4.76 mmol) in THF (5 mL) wasdropwise added to the mixture at 0° C. The reaction mixture was stirredat 0° C. for 2 hours. The solvent was removed under a reduced pressureand the residue was purified by column chromatography (5 to 10% EtOAc inhexanes) to afford methyl3-[2-(acetylthio)ethyl]-1H-indole-2-carboxylate (0.864 g, 63% for thethree steps) as a white solid: ¹H NMR (DMSO-d₆) 2.35 (s, 3H), 3.10–3.16(m, 2H), 3.28–3.35 (m, 2H), 3.92 (s, 3H), 7.13 (t, J=7.3 Hz, 1H), 7.31(t, J=7.5 Hz, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.77 (d, J=8.1 Hz, 1H).

3-(2-Mercaptoethyl)-1H-indole-2-carboxylic acid

To a deoxygenated solution of3-[2-(acetylthio)ethyl]-1H-indole-2-carboxylate (0.277 g, 1.0 mmol) inTHF (5 mL) was added deoxygenated 0.5 N KOH (10 mL), and the mixture wasstirred at room temperature under nitrogen for 20 hours. The volatilesolvent was removed under reduced pressure and the resulting aqueousmixture was poured into a mixture of EtOAc/1 N HCl (10 mL/10 mL). Theorganic layer was dried over Na₂SO₄ and concentrated. The crude materialwas purified by silica gel chromatography (EtOAc/hexanes, 1/1) to give0.150 g (0.68 mmol, 68% yield) of3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid as a white solid: ¹H NMR(CDCl₃) δ 1.49 (t, J=8.0 Hz, 1H), 2.91 (q, J=7.7 Hz, 2H), 3.48 (t, J=7.5Hz, 2H), 7.19 (ddd, J=1.3, 6.6, and 8.1 Hz, 1H), 7.38 (t, J=7.4 Hz, 1H),7.42 (d, J=8.1 Hz, 1H), 7.75 (d, J=8.1 Hz, 1H), 8.85 (s, 1H); ¹³C NMR(CDCl₃) □ 25.4, 29.5, 112.0, 120.7, 121.1, 122.4, 124.7, 126.6, 127.9,136.3, 167.0. Anal. Calcd. for C₁₁H₁₁NO₂S: C, 59.71; H, 5.01; N, 6.33;S, 14.49. Found: C, 59.58; H, 5.07; N, 6.25; S, 14.28.

Example 2 Preparation of 3-(2-mercaptoethyl)-1H-indole-2,7-dicarboxylicacid

By the methods previously outlined in Example 1 but using1,3,4,9-tetrahydro-1-oxo-pyrano[3,4-b]indole-8-carboxylic acid methylester (prepared from methyl 2-aminobenzoate by using the methodspreviously described in Arch. Pharm., 1987, 320, 1202–1209) was made3-(2-mercaptoethyl)-1H-indole-2,7-dicarboxylic acid: ¹H NMR (CD₃OD) δ2.82 (t, J=7.5 Hz, 2H), 3.43 (t, J=7.5 Hz, 2H), 7.23 (t, J=7.7 Hz, 1H),8.01 (d, J=7.5 Hz, 2H). Anal. Calcd. for C₁₂H₁₁NO₄S: C, 54.33; H, 4.18;N, 5.28; S, 12.09. Found: C, 54.14; H, 4.38; N, 5.20; S, 11.86.

Example 3 Preparation of1-[(3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid (SCHEME II) Methyl3-[2-(acetylthio)ethyl]-1-[[3-(methoxycarbonyl)-phenyl]-methyl]-1H-indole-2-carboxylate

To a solution of methyl 3-[2-(acetylthio)ethyl]-1H-indole-2-carboxylate(0.300 g, 1.1 mmol) in DMF (5 mL) was added sodium hydride (60%dispersion in mineral oil, 0.048 g, 1.2 mmol) at −10° C., and themixture was stirred at −10° C. for 15 minutes. To the mixture was addedmethyl 3-(bromomethyl)-benzoate (0.273 g, 1.2 mmol) at −15° C., and thereaction mixture was allowed to gradually come to room temperature andstirred overnight. To the mixture were added H₂O (20 mL) and EtOAc (20mL). The organic layer was dried over Na₂SO₄ and concentrated to givemethyl3-[2-(acetylthio)ethyl]-1-[[3-(methoxycarbonyl)phenyl]-methyl]-1H-indole-2-carboxylateas an oil (0.430 g). This material was used for the next reactionwithout further purification: ¹H NMR (DMSO-d₆) 2.32 (s, 3H), 3.10 (t,J=6.52 Hz, 2H), 3.31 (t, J=6.52 Hz, 2H), 3.80 (s, 3H), 3.83 (s, 3H),5.85 (s, 2H), 7.17–7.23 (m, 2H), 7.34 (t, J=7.7 Hz, 1H), 7.42 (t, J=7.6Hz, 1H), 7.56 (d, J=8.5 Hz, 1H), 7.66 (s, 1H), 7.80 (d, J=7.8 Hz, 1H),7.83 (d, J=8.1 Hz, 1H).

1-[(3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

To a solution of methyl3-[2-(acetylthio)ethyl]-1-[[3-(methoxycarbonyl)phenyl]methyl]-1H-indole-2-carboxylate

(0.430 g) in deoxygenated THF (7 mL) was added deoxygenated 1 N KOH (7mL). The mixture was stirred at room temperature under nitrogen for 24hours. The reaction mixture was washed with EtOAc and poured into 1 NHCl (25 mL) containing ice chips. The resulting white precipitate waswashed with H₂O and dried under vacuum to give 0.344 g of1-[(3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid (88% for the two steps) as a white solid: ¹H NMR (CD₃OD) δ 2.81 (t,J=7.6 Hz, 2H), 3.44 (t, J=7.6 Hz, 2H), 5.89 (s, 2H), 7.12–7.21 (m, 2H),7.27–7.36 (m, 2H), 7.39 (d, J=8.4 Hz, 1H), 7.71 (s, 1H), 7.76 (d, J=8.1Hz, 1H), 7.85 (d, J=7.7 Hz, 1H). Anal. Calcd. for C₁₉H₁₇NO₄S: C, 64.21;H, 4.82; N, 3.94; S, 9.02. Found: C, 64.44; H, 4.87; N, 3.86; S, 8.97.

Example 4 Preparation of1-[(2-bromo-5-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

By the methods previously outlined in Example 3 but using methyl4-bromo-3-(bromomethyl)benzoate was made1-[(2-bromo-5-carboxyphenyl)methyl]-3-(2-mercapto-ethyl)-1H-indole-2-carboxylicacid: ¹H NMR (DMSO-d₆) δ 2.35 (t, J=7.8 Hz, 1H), 2.79 (q, J=7.6 Hz, 2H),3.43 (t, J=7.6 Hz, 2H), 5.86 (s, 2H), 6.71 (d, J=1.9 Hz, 1H), 7.23 (t,J=7.4 Hz, 1H), 7.36 (t, J=7.7 Hz, 1H), 7.45 (d, J=8.5 Hz, 1H), 7.72 (dd,J=2.0, 8.2 Hz, 1H), 7.85 (d, J=8.3 Hz, 1H), 7.89 (d, J=8.0 Hz, 1H).Anal. Calcd. for C₁₉H₁₆BrNO₄S: C, 52.55; H, 3.71; N, 3.23; S, 7.38; Br,18.40. Found: C, 52.48; H, 3.80; N, 3.13, S, 7.30; Br, 18.27.

Example 5 Preparation of1-[(4-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

By the methods previously outlined in Example 3 but using methyl4-(bromomethyl)benzoate was made1-[(4-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid: ¹H NMR (CD₃OD) δ 2.82 (t, J=7.2 Hz, 2H), 3.45 (t, J=7.5 Hz, 2H),5.91 (s, 2H), 7.05 (d, J=8.0 Hz, 2H), 7.16 (t, J=7.3 Hz, 1H), 7.25–7.40(m, 2H), 7.77 (d, J=7.5 Hz, 1H), 7.89 (d, J=8.2 Hz, 2H). Anal. Calcd.for C₁₉H₇NO₄S: C, 64.21; H, 4.82; N, 3.94; S, 9.02. Found: C, 64.04; H,5.07; N, 3.84, S, 8.74.

Example 6 Preparation of3-(2-mercaptoethyl)-1-(phenylmethyl)-1H-indole-2-carboxylic acid

By the methods previously outlined in Example 3 but using benzyl bromidewas made 3-(2-mercaptoethyl)-1-(phenylmethyl)-1H-indole-2-carboxylicacid: ¹H NMR (CDCl₃) δ 1.48 (t, J=8.1 Hz, 1H), 2.89 (q, J=7.7 Hz, 2H),3.49 (t, J=7.6 Hz, 2H), 5.82 (s, 2H), 7.00–7.04 (m, 2H), 7.1–7.3 (m,6H), 7.77 (d, J=8.1 Hz, 1H); ¹³C NMR (CDCl₃) δ 25.6, 30.3, 48.3, 111.0,120.7, 121.1, 123.3, 126.2 (3C), 126.5, 126.8, 127.2, 128.6 (2C), 138.3,139.2, 167.0. Anal. Calcd. for C₁₈H₁₇NO₂SO.0.1H₂O: C, 69.03; H, 5.54; N,4.47; S, 10.24. Found: C, 68.88; H, 5.52; N, 4.50, S, 9.91.

Example 7 Preparation of1-[(2-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

By the methods previously outlined in Example 3 but using methyl2-(bromomethyl)benzoate was made1-[(2-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid: ¹H NMR (CDCl₃) δ 2.84 (t, J=7.6 Hz, 2H), 3.47 (t, J=7.6 Hz, 2H),6.10–6.20 (m, 2H), 6.21 (s, 2H), 7.16 (t, J=7.3 Hz, 1H), 7.20–7.33 (m,4H), 7.79 (d, J=8.2 Hz, 1H), 8.00–8.10 (m, 1H). Anal. Calcd. forC₁₉H₁₇NO₄S.0.25H₂O: C, 63.41; H, 4.90; N, 3.89; S, 8.91. Found: C,63.63; H, 4.94; N, 4.00, S, 8.54.

Example 8 Preparation of1-[[3-carboxy-5-(1,1-dimethylethyl)phenyl]methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

By the methods previously outlined in Example 3 but using methyl3-(bromomethyl)-5-(1,1-dimethylethyl)-benzoate was made1-[[3-carboxy-5-(1,1-dimethylethyl)phenyl]methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid: ¹H NMR (CD₃OD) δ 1.21 (s, 9H), 2.82 (t, J=7.5 Hz, 2H), 3.44 (t,J=7.6 Hz, 2H), 5.89 (s, 2H), 7.15 (t, J=7.9 Hz, 1H), 7.22 (s, 1H), 7.32(t, J=8.2 Hz, 1H), 7.42 (d, J=8.5 Hz, 1H), 7.52 (s, 1H), 7.76 (d, J=8.1Hz, 1H), 7.90 (s, 1H). Anal. Calcd. for C₂₃H₂₅NO₄S.0.1H₂O: C, 66.84; H,6.15; N, 3.39; S, 7.76. Found: C, 66.78; H, 6.17; N, 3.38, S, 7.60.

Example 9 Preparation of1-[(4-bromo-3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

By the methods previously outlined in Example 3 but using methyl2-bromo-5-(bromomethyl)-benzoate was made1-[(4-bromo-3-carboxyphenyl)methyl]-3-(2-mercapto-ethyl)-1H-indole-2-carboxylicacid: ¹H NMR (CD₃OD) δ 2.81 (t, J=7.6 Hz, 2H), 3.43 (t, J=7.6 Hz, 2H),5.82 (s, 2H), 6.95 (dd, J=8.3, 2.3 Hz, 1H) 7.16 (t, J=8.1 Hz, 1H), 7.32(t, J=8.1 Hz, 1H), 7.39 (d, J=8.4 Hz, 1H), 7.46 (d, J=2.2 Hz, 1H), 7.52(d, J=8.3 Hz, 1H), 7.76 (d, J=8.1 Hz, 1H). Anal. Calcd. forC₁₉H₁₆BrNO₄S.0.4H₂O: C, 51.69; H, 3.84; Br, 18.10; N, 3.17; S, 7.26.Found: C, 51.66; H, 3.89; Br, 18.12; N, 3.18, S, 7.13.

Example 10 Preparation of1-[(2-carboxy-5-methoxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

By the methods previously outlined in Example 3 but using methyl2-(bromomethyl)-4-methoxybenzoate was made1-[(2-carboxy-5-methoxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid: ¹H NMR (CD₃OD) δ 2.85 (t, J=7.3 Hz, 2H), 3.4–3.6 (m, 2H), 3.50 (s,3H), 5.57 (s, 1H), 6.21 (s, 2H), 6.7–6.9 (m, 1H), 7.1–7.4 (m, 3H), 7.80(d, J=7.8 Hz, 1H), 8.06 (d, J=8.8 Hz, 1H). Anal. Calcd. forC₂₀H₁₉NO₅S.0.25 H₂O: C, 61.60, H., 5.04, N. 3.59, S, 8.22. Found: C,61.47, H., 5.02, N, 3.74, S, 8.14.

Example 11 Preparation of3-(2-mercaptoethyl)-1-(2-phenylethyl)-1H-indole-2-carboxylic acid

By the methods previously outlined in Example 3 but using(2-iodoethyl)benzene was made3-(2-mercaptoethyl)-1-(2-phenylethyl)-1H-indole-2-carboxylic acid: ¹HNMR (CDCl₃) δ 1.50 (t, J=8.1 Hz, 1H) 2.90 (q, J=7.7 Hz, 2H) 3.09 (t,J=7.7 Hz, 2H) 3.48 (t, J=7.6 Hz, 2H) 4.78 (t, J=7.7 Hz, 2H) 7.1–7.4 (m,8H) 7.76 (d, J=8.1 Hz, 1H). Anal. Calcd. for C₁₉H₁₉NO₂S.0.1H₂O: C,69.74; H, 5.91; N, 4.28; S, 9.80. Found: C, 69.72; H, 5.98; N, 4.29; S,9.61.

Example 12 Preparation of3-(2-mercaptoethyl)-1-phenyl-1H-indole-2-carboxylic acid (SCHEME III)3-(2-acetylthioethyl)-1-phenyl-1H-indole-2-carboxylic acid, methyl ester

To a solution of methyl 3-[2-(acetylthio)ethyl]-1H-indole-2-carboxylate(0.364 g, 1.31 mmol), phenylboronic acid (0.239 g, 1.97 mmol), andcopper(II)acetate (0.237 g, 1.31 mmol) in dichloromethane (10 mL) wasadded pyridine (0.91 mL, 6.55 mmol), and the mixture was stirred at roomtemperature for 2 days. The reaction mixture was passed through a columnof silica gel (EtOAc/hexanes, 1:9) to afford3-(2-acetylthioethyl)-1-phenyl-1H-indole-2-carboxylic acid, methyl ester(0.153 g, 0.43 mmol, 33%) as a clear oil: ¹H NMR (CDCl₃) δ 2.37 (s, 3H),3.20–3.24 (m, 2H), 3.39–3.43 (m, 2H), 3.75 (s, 3H), 7.07 (d, J=8.2 Hz,1H), 7.20–7.32 (m, 4H), 7.42–7.53 (m, 3H), 7.87 (d, J=7.9 Hz, 1H).

3-(2-mercaptoethyl)-1-phenyl-1H-indole-2-carboxylic acid

To a deoxygenated solution of3-(2-acetylthioethyl)-1-phenyl-1H-indole-2-carboxylic acid, methyl esterin dioxane (5 mL) was added a deoxygenated solution of potassiumhydroxide (0.168 g, 3.0 mmol) in water (5 mL). The reaction mixture wasstirred overnight. Volatile solvent was removed under reduced pressureand the remaining mixture was poured into 1 N HCl (25 mL) containing icechips. The resulting white precipitate was washed with H₂O and driedunder vacuum to give 0.110 g of3-(2-mercaptoethyl)-1-phenyl-1H-indole-2-carboxylic acid (93% yield) asa white solid: ¹H NMR (CDCl₃) δ 1.52 (t, J=8.1 Hz, 1H), 2.91 (q, J=7.7Hz, 2H), 3.49 (t, J=7.6 Hz, 2H), 7.08 (d, J=8.2 Hz, 1H), 7.21 (t, J=7.5Hz, 1H), 7.27–7.33 (m, 3H), 7.42–7.53 (m, 3H), 7.78 (d, J=8.0 Hz, 1H).Anal. Calcd. for C₁₇H₁₅N₂O₂S.0.25 H₂O: C, 67.64, H, 5.18, N, 4.64, S,10.62. Found: C, 67.77, H, 5.15, N, 4.69, S, 10.62.

Example 13 Preparation of1-(3-carboxyphenyl)-3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid(SCHEME IV)3-Ethoxycarbonylmethyl-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid ethyl ester

A solution of (2-ethoxycarbonyl-indol-3-yl)-acetic acid ethyl ester (J.Med. Chem. 1991, 34, 1283–1292, 2.2 g, 8.0 mmol), methyl-3-bromobenzoate(5.16 g, 24 mmol), anhydrous powdered K₂CO₃ (2.2 g, 16 mmol), and copper(I) bromide (2.3 g, 16 mmol) in N-methylpyrrolidone (NMP, 10 mL) wasstirred at 170° C. overnight under inert atmosphere. The solvent wasthen removed by stream of nitrogen, and the residue was suspended inEtOAc (20 mL). The suspension was passed through a pad of silica gelusing EtOAc-hexanes (1:2) as an eluent. The filtrate was concentrated invacuo and purified by HPLC on LUNA 10μ silica column 25×250 mm with RIdetector to afford 2.4 g of3-ethoxycarbonylmethyl-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid ethyl ester as a white solid (73% yield): ¹H NMR (CDCl₃) δ 1.11 (t,J=7.3 Hz, 3H), 1.27 (t, J=7.1 Hz, 3H), 3.91 (s, 3H), 4.12–4.24 (m, 6H),7.01–7.06 (m, 1H), 7.17–7.32 (m, 2H), 7.51–7.62 (m, 2H), 7.69–7.75 (m,1H), 8.01–8.04 (m, 1H), 8.11–8.14 (m, 1H).

3-Carboxymethyl-1-(3-carboxy-phenyl)-1H-indole-2-carboxylic acid

To a solution of3-ethoxycarbonylmethyl-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid ethyl ester (1.31 g, 3.2 mmol) in THF-methanol (2:1, 25 mL) wasadded a solution of NaOH (0.64 g, 16.0 mmol) in H₂O (5 mL). The mixturewas stirred at room temperature overnight. The solvent was removed underreduced pressure and the residue was diluted with H₂O (50 mL) and washedwith diethyl ether (50 mL). The aqueous layer was acidified withconcentrated HCl to pH=1. The white precipitated was filtered off anddried in vacuo to afford 1.08 g of white solid (quantitative yield): ¹HNMR (DMSO-d6) δ 4.13 (s, 2H), 7.05 (d, 1H), 7.21 (t, 1H), 7.31 (t, 1H),7.62–7.71 (m, 2H), 7.76–7.82 (m, 2H), 8.01–8.05 (m, 1H); ¹³C NMR(DMSO-d6) 31.0, 111.1, 119.0, 121.2, 121.4, 126.2, 127.4, 127.8, 128.3,128.7, 129.9, 132.2, 138.9, 139.3, 162.6, 167.0, 172.5. Anal: Calcd forC₁₈H₁₃NO₆: C, 63.72; H, 3.86; N, 4.13. Found: C, 63.32; H, 4.08; N,3.97.

1-(3-Carboxy-phenyl)-3-ethoxycarbonylmethyl-1H-indole-2-carboxylic acid

To a solution of solution of3-carboxymethyl-1-(3-carboxy-phenyl)-1H-indole-2-carboxylic acid (0.743g, 2.2 mmol) in ethanol (5 mL) was added a drop of conc. H₂SO₄ and themixture was stirred at room temperature for 2.5 h. The reaction mixturewas diluted with water (50 mL) and extracted with EtOAc (50 mL). Theextract was washed with brine, dried over MgSO₄ and concentrated to give0.94 g of1-(3-carboxy-phenyl)-3-ethoxycarbonylmethyl-1H-indole-2-carboxylic acidas a yellow oil (94% yield): ¹H NMR (CDCl₃, 300 MHz) δ 1.17 (t, J=7.25Hz, 3H), 4.09 (q, J=7.05 Hz, 2H), 4.18 (s, 2H), 7.04 (d, J=8.39 Hz, 1H),7.20 (t, J=7.25 Hz, 1H), 7.30 (t, J=7.25 Hz, 1H), 7.71–7.60 (m, 2H),7.76–7.80 (m, 2H), 8.02 (d, J=7.06 Hz, 1H).

1-(3-Carboxy-phenyl)-3-(2-hydroxyethyl)-1H-indole-2-carboxylic acid

To a suspension of LiBH₄ (0.157 g, 7.2 mmol) in anhydrous DME (50 mL)was added a solution of1-(3-carboxy-phenyl)-3-ethoxycarbonylmethyl-1H-indole-2-carboxylic acidin DME containing 3% methanol (10 mL). The reaction mixture was heatedat reflux for 1 h and then allowed to cool to room temperature. Thereaction was quenched with 1 N HCl at 0° C. and extracted with EtOAc.The extract was dried over Na₂SO₄ and concentrated to afford 0.78 g of1-(3-carboxy-phenyl)-3-(2-hydroxyethyl)-1H-indole-2-carboxylic acid as ayellow oil (100% crude yield): ¹H NMR (CDCl₃, 300 MHz) δ 3.28 (t, J=6.87Hz, 2H), 3.69 (t, J=6.87 Hz, 2H), 7.03 (d, J=8.20 Hz, 1H); 7.18(t,J=7.25 Hz, 1H), 7.28 (t, J=7.25 Hz, 1H), 7.60–7.68 (m, 2H), 7.76–7.81(m, 2H), 8.02 (d, J=7.06 Hz, 1H).

3-(2-Hydroxy-ethyl)-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid methyl ester

To a solution of1-(3-carboxy-phenyl)-3-(2-hydroxyethyl)-1H-indole-2-carboxylic acid(0.40 g, 1.2 mmol) in methanol (5 mL) was dropwise added an ethersolution of diazomethane until persistent yellow color was observed. Thereaction mixture was concentrated to give 0.45 g of3-(2-Hydroxy-ethyl)-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid methyl ester as a yellow oil (100% crude yield): ¹H NMR (CDCl₃, 300MHz) δ 3.18 (t, J=6.67 Hz, 2H), 3.43 (s, 2H), 3.67 (s, 2H), 3.74 (t,J=6.67 Hz, 2H), 6.79 (d, J=8.20 Hz, 1H), 6.93–7.07 (m, 2H), 7.23–7.36(m, 2H); 7.52 (d, 7.82 Hz, 1H); 7.73(m, 1H); 7.88 (dt, J=8.97, 1.52 Hz,1H).

1-(3-Methoxycarbonyl-phenyl)-3-[2-(toluene-4-sulfonyloxy)-ethyl]-1H-indole-2-carboxylicacid methyl ester

A solution of3-(2-Hydroxy-ethyl)-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid methyl ester (0.41 g, 1.16 mmol), TsCl (0.23 g, 1.22 mmol),triethylamine (0.2 mL, 1.4 mmol) in dichloromethane (10 mL) was stirredat room temperature for 48 h. The solvent was removed under reducedpressure and the residue dissolved in EtOAc. The organic solution waswashed with 10% KHSO₄, dried over Na₂SO₄, and purified by silica gelchromatography (hexanes/EtOAc, 1/2) to give 0.37 g of1-(3-methoxycarbonyl-phenyl)-3-[2-(toluene-4-sulfonyloxy)-ethyl]-1H-indole-2-carboxylicacid methyl ester as a yellow oil (63% yield): ¹H NMR (CDCl₃, 300 MHz) δ2.37 (s, 3H), 3.52 (t, J=7.24 Hz, 2H), 3.67 (s, 3H), 3.92 (s, 3H), 4.34(t, J=7.06, 2H), 7.00 (d, J=8.24 Hz, 1H), 7.17–7.31(m, 4H), 7.44 (d,J=8.02 Hz, 1H), 7.56–7.67(m, 4H), 7.93 (t, J=1.72 Hz, 1H); 8.13 (dt,J=7.82, 1.34 Hz, 1H).

3-(2-Acetylsulfanyl-ethyl)-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid methyl ester

A solution of1-(3-methoxycarbonyl-phenyl)-3-[2-(toluene-4-sulfonyloxy)-ethyl]-1H-indole-2-carboxylicacid methyl ester (0.37 g, 0.73 mmol) and potassium thioacetate (0.092g, 0.8 mmol) in DMF (5 mL) was stirred at room temperature overnight.The reaction mixture was diluted with water and extracted with EtOAc.The organic extract was washed with saturated NaHCO₃, dried over Na₂SO₄,and concentrated to give 0.32 g of3-(2-acetylsulfanyl-ethyl)-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid methyl ester as a yellow oil (100% crude yield): ¹H NMR (CDCl₃, 300MHz) δ 2.36 (s, 3H), 3.20 (q, J=6.87 Hz, 2H), 3.40 (q, J=6.87 Hz, 2H),3.74 (s, 3H), 3.91 (s, 3H), 7.01 (d, J=7.82 Hz, 1H), 7.20–7.31 (m, 3H),7.44 (d, J=8.02 Hz, 1H), 7.48–7.52 (m, 1H), 7.59(t, J=7.82 Hz, 1H), 7.88(d, J=7.82 Hz, 1H), 7.97(t, J=1.53 Hz, 1H); 8.13 (dt, J=7.82, 1.34 Hz,1H).

1-(3-Carboxy-phenyl)-3-(2-mercapto-ethyl)-1H-indole-2-carboxylic acid

To a solution of3-(2-acetylsulfanyl-ethyl)-1-(3-methoxycarbonyl-phenyl)-1H-indole-2-carboxylicacid methyl ester (0.32 g, 0.73 mmol) in THF (10 mL) was dropwise added0.5 N KOH (10 mL) and the resulting mixture was stirred at roomtemperature overnight. The reaction acidified with 3 N HCl and theresulting white precipitate was washed with H₂O and dried under vacuumto give 0.22 g of a white solid (85% yield over 2 steps): ¹H NMR(DMSO-d6, 300 MHz) δ 2.81 (t, J=6 Hz, 2H), 3.37 (t, J=6 Hz, 2H), 7.02(d, J=9 Hz, 1H), 7.19 (t, J=6 Hz, 1H), 7.28 (t, J=6 Hz, 1H), 7.63 (m,2H), 7.77 (s, 1H), 7.83 (d, J=9 Hz, 1H), 8.01 (d, J=9 Hz, 1H); ¹³C NMR(DMSO-d6, 300 MHz) δ 25.18, 29.62, 111.11, 121.26, 123.93, 126.18,127.13, 127.28, 128.35, 129.10, 129.86, 132.24, 138.93, 139.48, 162.78,167.07. Anal. Calcd for C₁₈H₁₅NO₄S: C, 63.33; H, 4.43; N, 4.10; S, 9.39.Found: C, 63.03; H, 4.6; N, 4.09; S, 9.16.

Example 14 Preparation of1-[3-carboxy-5-(1,1-dimethylethyl)-phenyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid

By the methods previously outlined in Example 13 but using5-t-butyl-methyl-3-bromobenzoate in place of methyl-3-bromobenzoate.

Example 15 In Vitro Inhibition of NAALADase Activity

Various compounds of the invention were tested for in vitro inhibitionof NAALADase activity. The average IC₅₀ values of the tested compoundsare provided below in TABLE II.

TABLE II In Vitro Inhibition of NAALADase Activity Compound No. IC₅₀(nM) 1 1240 2 760 3 55.3 4 54.3 5 66.5 6 12100 7 18.5 8 35 9 104 10 1211 4740 12 44000

Protocol for Assaying In Vitro Inhibition of NAALADase Activity

The following were combined in assay tubes: 100 μL of 10 mM CoCl₂, 250μof 200 mM Tris chloride, 100 μL tissue, 100 μL of 10 mM NAALADaseinhibitor in Bakers H₂O, and Bakers H₂O to make a total volume of 950μL. Each assay tube was then incubated for 10 minutes in a 37 EC waterbath. 50 μL of 3-H—NAAG was then added to each assay tube and incubatedfor an additional 15 minutes in a 37 EC water bath. The assay wasstopped by adding 1.0 ml of 0.1 M sodium phosphate.

Glutamate released by the action of the NAALADase enzyme was separatedfrom the assay solution using an anion exchange resin. The resin wasequilibrated to 25 EC, 2.0 ml of the resin was added to a Pasteurpipette pre-loaded with a single glass bead, and each column was washedtwice with distilled H₂O. A column was placed over a scintillation vialand 200 μL of an assay sample was loaded onto the column. Afterdraining, glutamate was eluted using two 1.0 ml washes of 1 M formicacid. After addition of 10 ml of scintillation cocktail, each sample wascounted for 2 minutes on a scintillation counter.

Example 16 Effect of NAALADase Inhibition on TGF-β in In Vitro IschemiaModel

A NAALADase inhibitor, Compound C, was added to ischemia cell culturesto determine its effect on TGF-β levels during stroke. The experimentaldata, set forth in FIGS. 1 and 2, show increased concentrations ofTGF-β1 (FIG. 1) and TGF-β2 (FIG. 2) in ischemic cell cultures treatedwith Compound C. The results indicate that NAALADase inhibition promotesthe release of endogenous TGF-β's from glial cells, which in turnprovides neuroprotection for neighboring neurons.

TGF-β neutralizing antibodies were then added to the ischemic cellcultures. FIG. 3 shows that the TGF-β neutralizing antibodies blockedthe neuroprotective effect of Compound C in the in vitro ischemia model.By contrast, FIG. 4 shows that the addition of another growth factorantibody, FGF antibody, did not block the neuroprotective effect ofCompound C. The results indicate that NAALADase inhibition specificallyaffects TGF-β levels during stroke.

Example 17 Effect of NAALADase Inhibition on TGF-β in In Vivo IschemiaModel

The effect of TGF-β neutralizing antibodies on the neuroprotectiveeffect of Compound C was also studied in rats following MCAO. FIG. 6shows that treatment of MCAO rats with Compound C caused a significantrise in TGF-β1 levels during both occlusion and reperfusion, as assessedby microdialysis. The results indicate that NAALADase inhibitionprovides neuroprotection, at least in part, by regulating endogenousTGF-β's.

Additionally, FIG. 5 shows that TGF-β neutralizing antibodiessignificantly attenuated the neuroprotective effect of Compound C invivo. One of ordinary skill in the art can appreciate that theregulation of TGF-β's by NAALADase inhibitors may have implications notonly in stroke, but also in other diseases, disorders and conditionsincluding, without limitation, neurological diseases, psychiatricdiseases, demyelinating diseases, prostate cancer, inflammation,diabetes and angiogenesis.

Example 18 In Vivo Assay of NAALADase Inhibitors on Neuropathic Pain inSTZ Model

Male Sprague-Dawley rats (200–225 g) were rendered diabetic byintravenous administration of streptozotocin (“STZ”, 70 mg/kg inphosphate buffered saline). Diabetic animals were divided into fivegroups: one group receiving Compound A (10 mg/kg or 1 mg/kg), Compound D(10 mg/kg or 1 mg/kg) or vehicle. Another group of animals (non-STZtreated) served as non-diabetic controls. Drug/vehicle treatment wasstarted in diabetic animals 45 days post-STZ administration. STZ-induceddiabetic rats were tested for sensitivity to a heat source as soon asblood glucose levels rose to 320 mg/dl or above (30 days post STZ). Therats were then acclimated to a Hargreaves apparatus and thermalnociception was monitored using an infrared heat source directed intothe dorsal surface of the hindpaw, and the time taken for the animal toremove its paw noted to the nearest 0.1 seconds (see Hargreaves et al.,supra, for detailed experimental method). The intensity of the beamsource was adjusted such that the mean latency for control animals(non-STZ treated) was approximately 10 seconds. Each animal was tested 8times and the mean difference score (between mean non-diabetic controllatency and mean diabetic latency) are graphically presented in FIGS. 7Aand 7B. Diabetic rats displayed a hyperalgesia (shorter responselatency) compared to non-diabetic controls, starting 30 days post STZtreatment and progressively worsening in vehicle treated rats. Thishyperalgesic response was completely reversed in diabetic rats receivingtreatment with Compound D or A (10 mg/kg i.p. daily). Thus, the resultsshow that NAALADase inhibition attenuates neuropathic pain.

Example 19 In Vivo Assay of NAALADase Inhibitors on Neuropathic Pain inChronic Constriction Injury (“CCI”) Model

Sciatic nerve ligation, consisting of 4 ligatures tied loosely aroundthe sciatic nerve at 1 mm intervals proximal to the nerve trifurcation,was performed on rats. Following sciatic nerve ligation, the ratsexhibited a thermal hyperalgesia and allodynia. The rats were habituatedto a Hargreaves apparatus. An infrared heat source was directed onto thedorsal surface of each rat's hindpaws and the time taken for the rat towithdraw its paws was noted. The difference in scores between thelatency of the response for the paw on the operated side versus the pawon the unoperated control side was determined.

Compound C

The rats received either Compound C (50 mg/kg i.p. daily) or a vehiclestarting 10 days post surgery. Treatment with Compound C dramaticallynormalized the difference scores between the two paws compared to thecontinued hyperalgesic vehicle-treated controls. Normal (unoperated)rats had approximately equal latencies for both paws. This effect wassignificant starting at 11 days of drug treatment and persisted throughto the end of the study (for 21 days of daily dosing). The differencescores are graphically presented in FIG. 8. The results show thatNAALADase inhibition attenuates CCI-associated hyperalgesia.

Example 20 In Vivo Assay of NAALADase Inhibitors on Progression ofNeuropathic Pain in BB/W Models

Compounds D and A

Male BB/W rats (BRI, Mass) spontaneously develop a cell mediatedautoimmune destruction of pancreatic B cells, resulting in onset ofinsulin-dependent (Type I) diabetes (Guberski 1994). These rats havebeen characterized and shown to demonstrate neuropathies withaccompanying neural deficits such as fiber loss and degeneration,changes which are correlative with those seen in peripheral nerve ofhuman diabetic patients (Yagihasi 1997). This renders them valuable forexperimental trials of new compounds for future treatments of this majordisorder. In the present study, Compound D and Compound A were examinedfor their ability to alter the progression of diabetic neuropathy. Therats received daily injection of Compound D or Compound A (10 mg/kgi.p.) or vehicle, starting at the onset of diabetes (hyperglycemia) andup to 6 months thereafter. Another group of non-diabetic rats alsoreceiving vehicle were tested. All animals were continuously monitoredfor body weight, urine volume, blood sugar and glycated haemoglobin. Inthe first month of the study, all animals were tested for thermalnociception in a Hargreaves apparatus, weekly. After the first monththis was done biweekly and then monthly. The testing consists ofdirecting an infrared heat source onto the dorsal surface of the rathindpaw and noting the time taken for the animal to remove its paw (seeHargreaves et al., supra, for a description of the experimental method).Each animal was tested 8 times and the mean withdrawal latency noted.

The results are graphically presented in FIG. 11. The results show thatdiabetic rats displayed a hyperalgesia (shorter response latency)compared to non-diabetic controls. Diabetic drug-treated rats (bothCompound D and Compound A) displayed longer withdrawal latencies thandiabetic vehicle-treated rats, starting after 4 weeks of treatment andpersisting through the six months of treatment.

Nerve conduction velocity was also measured every two weeks through thefirst eight weeks of treatment and every month thereafter through to thesix months of treatment (see De Koning et al., Peptides, Vol. 8, No. 3,pp. 415–22 (1987) for a description of the experimental method). Theresults are graphically presented in FIG. 12. Diabetic animals generallyshowed a reduction in nerve conduction velocity compared to non-diabeticcontrols. However, diabetic animals receiving daily injections ofNAALADase inhibitor (either Compound D or Compound A at a dose of 10mg/kg) showed significantly less severe nerve conduction deficits thandid the diabetic controls receiving vehicle treatment. This was apparentstarting at 8 weeks of treatment and persisted to a similar degreethrough to the six month termination point of the study. Diabeticvehicles, on the other hand, showed a progressive deterioration in nerveconduction velocity from 6 to 16 weeks after start of vehicleadministration which was maintained through to six months.

Example 21 In Vivo Assay of NAALADase Inhibitors on Diabetic Neuropathyin STZ Model

Motor and sensory nerve conduction velocity was also measured inSTZ-diabetic animals after 4, 8 and 12 weeks of treatment (see De Koninget al., supra, for a description of the experimental method). Briefly,stimulating needle electrodes were inserted close to the sciatic andtibial nerves with recording electrodes being placed subcutaneously overthe distal foot muscles, in anesthetized rats. The results aregraphically presented in FIGS. 9A, 9B, 10A and 10B. Diabetic animalsreceiving vehicle showed a significant reduction in both motor andsensory nerve conduction compared to non-diabetic animals. Treatmentwith 10 mg/kg of Compound A daily for 4, 8 and 12 weeks all tended toimprove (increase) both motor and sensory nerve conduction velocities,with a significant improvement being observed after 12 weeks and 8 weeksfor motor and sensory nerve conduction velocity, respectively (FIGS. 9Aand 9B). The lower dose of Compound A tested (1 mg/kg) had similareffects. Treatment of animals with Compound D at either dose alsoincreased both motor and sensory nerve conduction velocities above thatof diabetic controls, significantly so after 12 weeks of treatment forthe 10 mg/kg treatment group (FIGS. 10A and 10B) and at the earlier timeperiods after treatment with the 1 mg/kg dose. Thus, the results showthat NAALADase inhibition alters the progression of diabetic neuropathy.

Example 22 In Vivo Assay of NAALADase Inhibitors on Reversal of DiabeticNeuropathy in STZ Models

General Method for STZ Model—Delayed Dosing

Rats (200–225 grams) were injected with STZ (70 mg/kg) into the tailvein. Diabetes (>350 mg/dl) was confirmed in all rats, 4 weeks after STZadministration. Rats were left untreated until 35–49 days after STZ.Compound D (1, 3, or 10 mg/kg), Compound E (10 mg/kg), or vehicle weredosed daily p.o. following confirmation of hyperalgesia and/or nerveconduction velocity deficits. In separate experiments, onset oftreatment was delayed until 60 to 90 days after STZ administration.Nerve conduction velocity or withdrawal response to thermal stimulationof hind paws was measured at intervals, usually bi-weekly for thermalresponse and monthly for nerve conduction velocity.

General Method for db/db Mice Study

Spontaneously diabetic mice (db/db mice) and non-diabetic littermateswere obtained from Jackson labs. Mice were left untreated until 7–8months of age (or after 4–5 months of chronic diabetes) and then doseddaily with Compound F (1 mg/kg) p.o. Nerve conduction velocity wasmeasured prior to the onset and after eight weeks of treatment.

Nerve Conduction Velocity Measurements

Sensory and motor nerve conduction velocities were evaluated using themethod of De Koning and Gispen (Peptides 8: 415–422, 1987).Electrophysiological evaluation was carried out within one hour ofdosing. Animals were anesthetized with isoflurane and stimulating needleelectrodes were inserted close to the sciatic nerve at the sciatic notchand the tibial nerve near the ankle. Recording electrodes were placedover the foot muscles. Stimuli were applied and responses recorded.Motor and sensory nerve conduction velocities were calculated bymeasuring the distance between the sciatic notch and ankle sites, andthe latency between the M-wave and the H-reflex.

Thermal Hyperalgesia

Animals were acclimated to the apparatus for at least 5 min. Aninfra-red source was placed under below the plantar surface of the rathind-paw. The intensity of the source was adjusted so that latency fornormal rats was about 10 secs. Animals were tested for thermal responselatency according to the method of Hargreaves et al (Pain 77–88, 1988).Each animal was tested 8 times (4 each hind limb) and the latency ofresponse recorded automatically to nearest 0.1 sec. An average of thelast 4 measurements for each paw was calculated (8 total measurements)and noted for each rat.

FIG. 20 shows the effect of NAALADase inhibitor (Compound D and CompoundE) treatment on neuropathic pain abnormalities in STZ-diabetic rats. Allrats showed apparent hyperalgesia compared to non-diabetic rats prior toNAALADase inhibitor treatment (5 weeks post STZ). However, within twoweeks of treatment, neuropathic hyperalgesia was reversed towards normalin both NAALADase inhibitor treated groups. This reversal persistedthroughout the subsequent hypoalgesic phase usually seen in prolongeddiabetic-STZ rats, with a reduced hypoalgesic phase displayed inNAALADase treated rats.

FIG. 21 shows the motor nerve conduction velocity measurements in STZdiabetic rats and non-diabetic controls prior to and at time periodsafter NAALADase inhibitor treatment. Within 8 weeks of dosing, bothNAALADase inhibitors Compound D and Compound E reversed the motor nerveconduction velocity towards normal (non-diabetic values). This effectpersisted through 12 weeks of treatment.

FIG. 22 shows sensory nerve conduction velocity deficits, similarlytested. NAALADAse inhibitor treatment similarly reversed sensory nerveconduction velocity deficits, significantly so after only 2 weeks oftreatment.

FIG. 23 shows neuropathic pain abnormalities in another experiment wheretreatment with lower doses (1 and 3 mg/kg) of the NAALADase inhibitorCompound D was initiated after 7 weeks of STZ treatment. Significantreduction in pain abnormalities were again apparent with both doses ofCompound D.

FIGS. 24 and 25 show sensory and motor nerve conduction velocity,respectively, in these chronically diabetic STZ rats treated with thelower doses of Compound D. Sensory nerve conduction was significantlyimproved towards normal within 4 weeks of treatment whereas motor nerveconduction remained unimproved by these low doses, even 8 weeks afterdosing.

FIGS. 26 and 27 show sensory and motor nerve conduction velocitymeasurements generated from an external CRO in a similar chronicallydiabetic STZ model, where rats were left untreated until 60 days afterSTZ treatment. Partial reversal of both deficits was again produced byCompound D treatment. FIG. 28 shows the same where treatment was delayedyet further, until 90 days after STZ.

FIG. 29 shows nerve conduction velocity measurements from a geneticmouse model of diabetes, at 6–7 months of age (after about 4 months ofchronic diabetes). A significant impairment in sensory NCV was apparentat this time. FIG. 30 shows the nerve conduction velocity in these miceafter 8 weeks of treatment with another, more potent NAALADase inhibitoradministered at 1 mg/kg daily. Significant improvement in the sensorynerve conduction was apparent following drug treatment.

Example 23 Effect of NAALADase Inhibitors on Onset of ALS

The effect of NAALADase inhibitors on the onset of ALS was tested usingthe transgenic mice model of familial Amyotrophic Lateral Sclerosis(FALS), which is detailed in Gurney, M., Annals of Neurology (1996)39:147–157, and otherwise well known in the art. One month oldtransgenic G1H mice were treated with daily intraperitoneal injectionsof a vehicle (50 mM HEPES-buffered saline) or a NAALADase inhibitor (50mg/kg Compound A). Clinical symptoms of the mice were monitored daily.The onset of clinical disease was scored by examining each mouse for itsshaking of limbs when suspended in the air by its tail, cross spread ofspinal reflexes, hindlimb paralysis, body weight and wheel runningactivity.

The results, set forth below in TABLE III, show that disease onset wasdelayed in mice treated with a NAALADase inhibitor.

TABLE III EFFECT OF NAALADASE INHIBITOR ON ONSET OF CLINICAL DISEASEDISEASE ONSET DISEASE ONSET FOR COMPOUND A FOR VEHICLE TREATED MICETREATED MICE STUDY (days) (days) DIFFERENCE Study 1 221 189 32 Study 2166 141 25

Example 24 Effect of NAALADase Inhibitor on ALS Survival and ClinicalSymptoms

The effect of NAALADase inhibitors on ALS survival and clinical symptomswas tested using again the transgenic mice model of FALS. One month oldtransgenic G1H mice were treated daily with a vehicle (50 mMHEPES-buffered saline) or a NAALADase inhibitor (30 mg/kg Compound B)p.o. (by oral administration). Clinical symptoms of the mice weremonitored twice a week. Such symptoms included shaking of limbs, gait,dragging of hind limbs, crossing of limbs, righting reflex andmortality. Gait and crossing of limbs were graded on an arbitrary scaleranging from 0 to 3, with 0 representing most normal and 3 representingleast normal, e.g. severest difficulty in walking or crossing limbs.Righting reflex was measured by the time (seconds) it took the mice toright themselves when placed on their sides on a flat surface.

The results, set forth in FIGS. 13–19, show that survival was prolongedand clinical symptoms were attenuated in mice treated with a NAALADaseinhibitor.

Example 25 Protective Effect of NAALADase Inhibitors in Experimental RatGlaucoma

Experimental Protocol

All experiments complied with the Association for Research in Vision andOphthalmology Statement for the Use of Animals in Ophthalmic and VisionResearch. 82 male Brown Norway rats (Rattus norvegicus), each weighingapproximately 250 gm, were treated using procedures approved by theAnimal Care Committee of the Johns Hopkins University School ofMedicine. The rats were housed with a 12 hour light/12 hour dark cycleand fed ad libitum.

EXPERIMENTAL GLAUCOMA: Unilateral elevation of intraocular pressure(“IOP”) was produced in 56 rats by microinjection of hypertonic salineinto episcleral veins, following procedures described in Morrison, J. etal., IOVS (March 1998) 39:526–531. Beginning on the day of IOPelevation, the rats were treated daily with intraperitoneal injectionsof either a vehicle (23 rats with 50 mM HEPES-buffered saline) or aNAALADase inhibitor (11 rats with 10 mg/kg of Compound A and 22 ratswith 10 mg/kg of Compound B). 11 saline treated rats, 11 Compound Atreated rats and 11 Compound B treated rats were sacrificed at 8 weeks,and the remaining rats at 12 weeks, after initial IOP elevation.

OPTIC NERVE TRANSECTION: The optic nerve was transected unilaterally in26 rats under intraperitoneal pentobarbital anesthesia. The conjunctivawas opened with scissors and the optic nerve exposed by traction onextraocular muscles. The transection was performed with microscissors 5mm posterior to the globe, with specific attention to avoidance ofinjury to major ocular blood vessels. Immediately after transection, theretina was examined ophthalmoscopically to assure that the retinalarterial blood supply was not disrupted. The conjunctiva was closed withabsorbable suture and the eye dressed with antibiotic ointment.Beginning on the day of transection, the rats were treated daily withintraperitoneal injections of either a vehicle (9 rats with 50 mMHEPES-buffered saline) or a NAALADase inhibitor (8 rats with 10 mg/kg ofCompound A and 9 rats with 10 mg/kg of Compound B). 5 saline treatedrats, 3 Compound A treated rats and 4 Compound B treated rats weresacrificed at 2 weeks, and the remaining rats at 4 weeks, aftertransection.

OPTIC NERVE COUNTING: The rats were sacrificed by exsanguination underdeep pentobarbital anesthesia. They were perfused through the heart with2% paraformaldehyde/2% glutaraldehyde in 0.1 M phosphate buffer, pH 7.2,and the eyes with attached optic nerves were removed. A cross-section ofthe optic nerve from both experimental (glaucoma or transection) andcontrol eyes was removed 1.5 mm posterior to the globe, 1 mm inthickness, and post-fixed in 2% osmium tetroxide in buffer. These wereprocessed into epoxy resin, sectioned at 1 micron and stained withtoluidine blue.

The area of the optic nerve cross-section was measured by outlining itsouter border at 10× magnification on an image analysis system (UniversalImaging Corp., Westchester, Pa.) with Synsys digital camera andMetamorph software. Three area measurements were taken and the meanvalue was determined. To measure the density and fiber diameterdistributions, images were captured with a 100× phase contrast objectivefrom 10 different areas of each nerve. These were edited to eliminatenon-neural objects and the size of each axon internal to the myelinsheath (its minimum diameter) and the density of axons/square mm werecalculated for each image and nerve. The mean density was multiplied bytotal nerve area to yield fiber number for each nerve. The total fibernumber in glaucoma or transection nerves was compared to the normal,fellow eye of each rat to yield a percent loss value. The number ofaxons counted among the 10 images was an approximately 20% sample of the80–90,000 axons in normal rat nerves. The person measuring axon numberwas masked to the protocol conducted on the nerves.

Results

EXPERIMENTAL GLAUCOMA: The mean fiber percent difference in thesaline-treated, control rats was significantly lower in their glaucomaeyes compared to their normal eyes, with a mean fiber loss of14.44±5.75% (n=11 rats; TABLE IV) in the 8 week follow-up group, and8.15±7.84% in the 12 week follow-up group (n=12 rats; TABLE V).

By contrast, there was no significant loss of fibers in either the 8week or 12 week NAALADase inhibitor-treated rats. The mean percent fiberloss in each NAALADase inhibitor-treated group was statistically lessthan the loss in the saline-treated, control groups (at 8 weeks, p=0.05for Compound A and p=0.02 for Compound B).

TABLE IV EXPERIMENTAL GLAUCOMA RESULTS IOP INTEGRAL FIBER PERCENT 8 WEEKDIFFERENCE ± NUMBER ± DIFFER- GROUP N SEM SEM ENCE ± SEM Compound A 11 85 ± 37.5 79156 ± 2436 −1.82 ± 2.92 * Compound B 11 116 ± 33.2 80785 ±2121 −0.82 ± 2.97 ** Control 11 104 ± 26.4 68295 ± 4617 14.44 ± 5.75

TABLE V EXPERIMENTAL GLAUCOMA RESULTS 12 WEEK IOP INTEGRAL PERCENT GROUPN DIFFERENCE FIBER NUMBER DIFFERENCE Compound B 11 109 ± 45.2 90504 ±1718 −3.21 ± 2.86  Control 12 158 ± 66.5 79827 ± 6783 8.15 ± 7.84

IOP Integral Difference=difference IOP exposure between glaucoma eye andnormal eye in each rat (mm Hg—days).

Percent Difference=mean percent difference in fiber number betweenglaucoma and normal eye in each rat (positive value indicates fewerfibers in the glaucoma eye).

Differences in IOP Integral Difference are not significant (p>0.05).

Differences in Percent Difference between drug-treated andsaline-treated, control rats at 8 weeks post insult are significant(p=0.05* and p=0.02**).

OPTIC NERVE TRANSECTION: The experimental transection data suggest aslowing or rescue of ultimate RGC death in rats treated with NAALADaseinhibitors at 2 weeks after transection. At 2 weeks after transection,both drug-treated groups had more remaining RGC axons than did thesaline-treated, control group, judged either by absolute number offibers or percent difference between transected eye and normal eye ineach rat (TABLE VI). Rats treated with Compound A and Compound B had,respectively, 3 times and twice as many remaining axons as thesaline-treated rats. All or nearly all RGC die within the first 2 monthsafter transection, regardless of any pharmacological treatment. Thus, by4 weeks after transection, more than 80% of RGC axons were gone in allgroups (TABLE VII). At 4 weeks after transection, there were nosignificant differences between the drug-treated rats and thesaline-treated rats.

TABLE VI OPTIC NERVE TRANSECTION 2 WEEKS FIBER NUM- PERCENT SURVIVAL NBER ± SEM DIFFERENCE ± SEM Compound A 3 26,426 ± 13,293 65.3 ± 17.8Compound B 4 19,550 ± 5,091  75.3 ± 6.6  Control 5 8,220 ± 4,668 90.2 ±5.35

TABLE VII OPTIC NERVE TRANSECTION 4 WEEKS FIBER NUM- PERCENT SURVIVAL NBER ± SEM DIFFERENCE ± SEM Compound A 5 13,599 ± 3,519 82.4 ± 4.0Compound B 5  5,162 ± 2,509 93.4 ± 3.1 Control 4 10,449 ± 3,648 86.9 ±4.7

Percent Difference=mean percent difference in fiber number betweenglaucoma and normal eye in each rat (positive value indicates fewerfibers in the glaucoma eye).

Differences in Percent Difference between drug-treated andsaline-treated, control rats are not statistically significant (p=0.05).

Example 26 Efficacy of NAALADase Inhibitors in Treating RetinalDisorders

Four (4) groups of rats received daily insulin injections to maintaintheir glucose levels at about 350 mg/dl. Starting at the onset ofhyperglycemia, NAALADase inhibitor 2-(3-sulfanylpropyl)-pentanedioicacid was administered daily for 6 months to one group of BB/W rats at adose of 10 mg/kg and to a second group of BB/W rats at a dose of 30mg/kg. A third group of BB/W rats and a fourth group of non-diabeticrats received daily vehicle treatment (50 mM Hepes buffered saline).

After six (6) months of NAALADase inhibitor or vehicle treatment, therats were sacrificed and their eyes were removed. From each rat, one eyewas processed for elastase digest while the other eye was processed fortransmission electron microscopy (TEM) and basement membrane (BM)thickness.

Elastase Digests

Retinal digests were prepared using elastase on retinas as described inLayer, N., Invest Ophthalmol Vis Sci (1993) 34:2097. Eyes were removedfrom recently killed BB/W rats (n=25) and age-matched transgeniccontrols (n=10). The retinas (n=35) were fixed at room temperature byimmersing the whole eye (slit at limbus) in 4% (w/v) paraformaldehyde in50 mmol/L Na—K phosphate buffer with 8% sucrose. The fixed retinas wererinsed in deionized water and were incubated for 3 minutes in a 37° C.agitating water bath in 40 units/mL elastase in Na—K phosphate bufferwith 150 mmol/L NaCl and 5 mmol/L ethylenediamine tetraacetic acid(EDTA), pH 6.5. The tissues were washed overnight in 100 mmol/L Tris-HCL(pH 8.5) and then transferred to deionized water for removal of theloosened vitreous and digested neural elements by gentle agitation usingthe sides of closed forceps and the sides and ends of very fine brushes.After all loose tissues were removed, the retinas were incubated oncemore in fresh enzyme for 3 minutes and then subjected to a secondovernight wash at room temperature in Tns-HCl buffer. On the third day,the retinas were again transferred to deionized water for additionalremoval of digested neural elements. The vascular network that wascompletely free of nonvascular elements was mounted flat by flotation inCa²⁺ and Mg²⁺ free Dulbecco's PBS on siliconized slides (#S1308, Oncor,Gaithersburg, Md.). After air drying in a dust free environment, themounts of the retinal microvasculature were stained using periodic acidSchiff reaction and hematoxylin counterstaining, as described in Luna,L., ed. Manual of Histologic Staining Methods of the Armed ForcesInstitute of Pathology (1968) McGraw-Hill, New York, N.Y. Thepreparations were then examined by light microscopy and photographed.

Endothelial/Pericyte (E/P) Ratios

The stained and intact retinal whole mounts were coded and subsequentcounting was done masked, as described in Cuthbertson, R., InvestOphthalmol Vis Sci. (1986) 27:1659–1664).

Ten fields at ×100 magnification were counted for endothelial andpericyte cells using previously described morphologic criteria (seeKuwabara, T., Arch Ophthalmol. (1960) 64:904–911). In every sample, atleast 200 cells were counted from the mid zone of the retina. Meanvalues for endothelial cell/pericyte (E/P) ratios were initiallycalculated in 3 retinas from each of the four (4) groups of rats.

Evaluation of BM Thickness

Each eye was fixed in 4% glutaraldehyde and dissected free of sclera andchoroids, then trimmed and postfixed in 1% osmium tetroxide. Afterdehydration and embedding, thin sections were stained with uranylacetate and lead citrate. Initially, BM thickness of retinal capillariesfrom 3 non-diabetic rats receiving vehicle, 3 diabetic animals receiving10 mg/kg 2-(3-sulfanylpropyl)-pentanedioic acid, and 3 diabetic ratsreceiving 30 mg/kg 2-(3-sulfanylpropyl)-pentanedioic acid were comparedwith 3 diabetic rats receiving a vehicle. At least 10 capillaries pereye from the inner nuclear and plexiform layers were photographed at amagnification of 10,000×. Exact magnification was determined for eachset of negatives with a 28,800 line/inch calibration grid. Negativeswere enlarged 3×. Measurements, to the nearest 0.25 mm, were made of thebasement membrane surrounding the endothelial cell and were takenperpendicular to the plane of the basement membrane, as described inBendayan, M., J. Electron Microsc Techn (1984) 1:243–270; and Gunderson,J. Microscopy (1980) 121:65–73). At least 20 measurements were taken foreach capillary and the BM thickness was expressed as an average of 20measurements.

Statistical Analysis

Statistical analysis for comparison among groups was performed using oneway analysis of variance (ANOVA) and Student's t test. Significance wasdefined as a value of p<0.05. Values were reported as mean±standarderrors from the mean (SEM), unless otherwise noted.

Results of Elastase Digest Preparations and E/P Ratios

In intact whole mounts of retinal digests the endothelial cell nuclei,seen medially within the vessel wall, were large, oval, pale stainingand protruded lumenally. Pericyte nuclei, seen more laterally, were darkstaining, small, round and protruded prominently away from the vesselwall. E/P counts were taken from mid zones of the retinas. The attachedfigures show 27,000× magnified photographs of retinal blood vessels froma control, non-diabetic rat (FIG. 31), from a control, diabetic ratafter six (6) months of treatment with a vehicle (FIG. 32), and from adiabetic rat after six (6) months of treatment with NAALADase inhibitor2-(3-sulfanylpropyl)-pentanedioic acid (FIG. 33). In the figures, “BM”refers to basement membrane, “EC” refers to endothelial cell, and “L”refers to vessel lumen.

NAALADase inhibition had no effect on blood glucose or body weight. Sixmonth high dose (30 mg/kg) treatment with2-(3-sulfanylpropyl)-pentanedioic acid resulted in a 29.0% reduction inBM thickness (diabetic vehicle=101.0±14.81 nm and diabeticNAALADase₃₀=71.7±4.07 nm), while treatment with the low dose resulted inan 18.5% decrease in BM thickness (NAALADase₁₀=82.3±4.07 nm). This wasaccompanied by a 37% reduction of E/P ratios in rats treated with thehigh dose 2-(3-sulfanylpropyl)-pentanedioic acid (diabeticvehicle=3.0±0.3 and NAALADase₃₀=1.9±0.4), while treatment with low doseresulted in a 20% reduction of the same cell ratios(NAALADase₁₀=2.4±0.5). See TABLE VIII.

TABLE VIII BM THICKNESS E/P RATIO RAT GROUP (nm) ± SD (n = 3) (n = 8–10)NON-DIABETIC CONTROLS 56.3 ± 4.78  1.7 ± 0.07 DIABETIC VEHICLE   101 ±14.81 3.0 ± 0.3 DIABETIC 30 MG/KG 71.7 ± 4.07 1.9 ± 0.4 NAAALADASEINHIBITOR DIABETIC 10 MG/KG 82.3 ± 4.07 2.4 ± 0.5 NAALADASE INHIBITORConclusions

The BB/W rats demonstrated an early change typically associated withdiabetic retinopathy (pericyte loss and basement membrane thickening)but did not show significant numbers of microanuerysms also typical ofdiabetic retinopathy or areas of acellular capillaries usually seen inmore advanced disease. The retinopathy observed in BB/W has beenpreviously characterized in Chakrabarti, Diabetes (1989) 38:1181–1186.

The results show that treatment with a NAALADase inhibitor causesimprovement in retinal pathology of diabetic rats. Specifically, theNAALADase inhibitor prevented pericyte loss and basement membranethickening in retinal vessels.

Example 27 Neuroprotective Effect of NAALADase Inhibitors in TransgenicMouse Model of Huntington's Disease

Behavioral Testing (Rotarod)

Transgenic HD mice of the N171-82Q strain and non-transgenic littermateswere treated with NAALADase inhibitor Compound B (30 mg/kg) or a vehiclefrom 10 weeks of age. The mice were placed on a rotating rod(“rotarod”). The length of time at which the mouse fell off the rotarodwas recorded as a measure of motor coordination. FIG. 34 shows thattransgenic HD mice treated with Compound B stayed longer on the rotarodthan similar transgenic HD mice treated with a vehicle. The treatmentwith Compound B had no effect on the rotarod performance of normalnon-HD mice.

The total distance traveled by the mice was also recorded as a measureof overall locomotion. FIG. 35 shows that while the vehicle treated HDmice demonstrated the lowest mean locomotor score, the treatment withNAALADase inhibitor had no apparent effect on overall locomotion.

Survival

The effects of Compound B and vehicle on the survival of transgenic HDmice (N171-82Q) were evaluated. Thirteen mice (six male and sevenfemale) were assigned to the Compound B treatment group, and fourteenmice (six male and eight female) were assigned to the vehicle treatmentgroup. Treatment was continued until all the mice died.

FIG. 36 shows the survival distributions over time by treatment group.The median survival time is 184 days for the Compound B treatment group,and 158.5 days for the vehicle treatment group. Although the Compound Btreatment group had a longer median survival time than the vehicletreatment group, the difference is not statistically significant(p-value=0.07).

FIGS. 37A and 37B show the survival distributions over time by treatmentgroup and sex. When analyzing the results specific to sex, female micetreated with Compound B had significantly prolonged survival time(p-value=0.03) compared to their vehicle treated counterparts. Withinthe vehicle treatment group, the males have better survival times thanthe females although this trend was not observed in the Compound Btreatment group. The data suggest that sex may influence survivaldistributions over time.

Example 28

A patient is suffering from any disease, disorder or condition whereNAALADase levels are altered, including any of the diseases, disordersor conditions described above. The patient may then be administered aneffective amount of a compound of the invention. It is expected thatafter such treatment, the patient would not suffer any significantinjury due to, would be protected from further injury due to, or wouldrecover from the disease, disorder or condition.

All publications, patents and patent applications identified above areherein incorporated by reference, as though set forth herein in full.

The invention being thus described, it will be apparent to those skilledin the art that the same may be varied in many ways without departingfrom the spirit and scope of the invention. Such variations are includedwithin the scope of the following claims.

1. A compound of formula I

or a pharmaceutically acceptable salts, hydrates or optical isomers ofsaid compound, wherein: A¹, A², A³ and A⁴ are independently hydrogen,C₁–C₉ alkyl, C₂–C₉ alkenyl, C₂–C₉ alkynyl, aryl, heteroaryl, carbocycle,heterocycle, C₁–C₉ alkoxy, C₂–C₉ alkenyloxy, phenoxy, benzyloxy,hydroxy, halo, nitro, cyano, isocyano, —COOR⁶, —COR⁶, —NR⁶R⁷, —SR⁶,—SOR⁶, —SO₂R⁶, —SO₂(OR⁶), —(C═O)NR⁶R⁷, —(C═O)NR⁶(CH₂)_(n)COOH,—NR⁶(C═O)R⁷ or —(CH₂)_(n)COOH, or any adjacent two of A¹, A², A³ and A⁴form with the benzene ring a fused ring that is saturated orunsaturated, aromatic or non-aromatic, and carbocyclic or heterocyclic,said heterocyclic ring containing 1 or 2 oxygen, nitrogen and/or sulfurheteroatom(s); n is 1–3; R, R¹, R², R³, R⁴, R⁵, R⁶ and R⁷ areindependently hydrogen, carboxy, C₁–C₉ alkyl, C₂–C₉ alkenyl, C₂–C₉alkynyl, aryl, heteroaryl, carbocycle or heterocycle; and said alkyl,alkenyl, alkynyl, aryl, heteroaryl, carbocycle, heterocycle, alkoxy,alkenyloxy, phenoxy, benzyloxy and fused ring are independentlyunsubstituted or substituted with one or more substituent(s).
 2. Thecompound of claim 1, wherein: A¹, A², A³ and A⁴ are independentlyhydrogen or —COOH; R¹, R², R³ and R⁴ are each hydrogen; and R⁵ ishydrogen, phenyl, benzyl or phenylethyl, wherein said phenyl, benzyl andphenylethyl are independently unsubstituted or substituted with one ormore substituent(s).
 3. The compound of claim 2, wherein R⁵ is benzylsubstituted with one or more substituent(s) independently selected fromthe group consisting of carboxy, halo, C₁–C₄ alkyl and C₁–C₄ alkoxy. 4.The compound of claim 1, wherein said compound is selected from thegroup consisting of: 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid;3-(2-mercaptoethyl)-1H-indole-2,7-dicarboxylic acid;1-[(3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(2-bromo-5-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(4-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid; 3-(2-mercaptoethyl)-1-(phenylmethyl)-1H-indole-2-carboxylic acid;1-[(2-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[[3-carboxy-5-(1,1-dimethylethyl)phenyl]methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(4-bromo-3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(2-carboxy-5-methoxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid; 3-(2-mercaptoethyl)-1-phenyl-1H-indole-2-carboxylic acid;3-(2-mercaptoethyl)-1-(2-phenylethyl)-1H-indole-2-carboxylic acid;1-(3-carboxyphenyl)-3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid; and1-[3-carboxy-5-(1,1-dimethylethyl)phenyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid.
 5. The compound of claim 1, wherein said compound is an enantiomeror an enantiomer-enriched mixture.
 6. A pharmaceutical compositioncomprising: (i) an effective amount of a compound of claim 1; and (ii) apharmaceutically acceptable carrier.
 7. The pharmaceutical compositionof claim 6, wherein said the compound of claim 1 is selected from thegroup consisting of: 3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid;3-(2-mercaptoethyl)-1H-indole-2,7-dicarboxylic acid;1-[(3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(2-bromo-5-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(4-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid; 3-(2-mercaptoethyl)-1-(phenylmethyl)-1H-indole-2-carboxylic acid;1-[(2-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[[3-carboxy-5-(1,1-dimethylethyl)phenyl]methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(4-bromo-3-carboxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid;1-[(2-carboxy-5-methoxyphenyl)methyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid; 3-(2-mercaptoethyl)-1-phenyl-1H-indole-2-carboxylic acid;3-(2-mercaptoethyl)-1-(2-phenylethyl)-1H-indole-2-carboxylic acid;1-(3-carboxyphenyl)-3-(2-mercaptoethyl)-1H-indole-2-carboxylic acid; and1-[3-carboxy-5-(1,1-dimethylethyl)phenyl]-3-(2-mercaptoethyl)-1H-indole-2-carboxylicacid.
 8. The pharmaceutical composition of claim 6, wherein: A¹, A², A³and A⁴ are independently hydrogen or —COOH; R¹, R², R³ and R⁴ are eachhydrogen; and R⁵ is hydrogen, phenyl, benzyl or phenylethyl, whereinsaid phenyl, benzyl or phenylethyl are independently substituted withone or more substituent(s).
 9. The pharmaceutical composition of claim8, wherein R⁵ is benzyl substituted with one or more substituent(s)independently selected from the group consisting of carboxy, halo, C₁–C₄alkyl and C₁–C₄ alkoxy.
 10. The pharmaceutical composition of claim 6,wherein the compound is an enantiomer or an enantiomer-enriched mixture.